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Chemical Space of DNA-Encoded Libraries Miniperspective Raphael M. Franzini* and Cassie Randolph Department of Medicinal Chemistry, College of Pharmacy, University of Utah, 30 S 2000 E, Salt Lake City, Utah 84112, United States S Supporting Information *
ABSTRACT: In recent years, DNA-encoded chemical libraries (DECLs) have attracted considerable attention as a potential discovery tool in drug development. Screening encoded libraries may offer advantages over conventional hit discovery approaches and has the potential to complement such methods in pharmaceutical research. As a result of the increased application of encoded libraries in drug discovery, a growing number of hit compounds are emerging in scientific literature. In this review we evaluate reported encoded libraryderived structures and identify general trends of these compounds in relation to library design parameters. We in particular emphasize the combinatorial nature of these libraries. Generally, the reported molecules demonstrate the ability of this technology to afford hits suitable for further lead development, and on the basis of them, we derive guidelines for DECL design.
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INTRODUCTION One of the fundamental steps of drug development is the identification of molecules that bind to a target biomacromolecule and exert a desired pharmaceutical effect. The discovery of such hits (i.e., initially identified compounds before optimization) is challenging. A common approach to identify hit molecules is to screen large compound libraries one member at a time using a suitable bioassay readout (i.e., highthroughput screening, HTS). This approach has provided countless leads for drug development.1 However, HTS relies on expensive compound libraries and sophisticated robotic equipment which is often unavailable to researchers in academia or at small companies. In stark contrast, the discovery of therapeutic biomolecules (e.g., antibodies) by display technologies (e.g., phage display, yeast display, mRNA display, ribosome display, SELEX) is generally rapid and inexpensive. Phage display for example can provide protein-binding peptides or antibodies within a few weeks using standard laboratory equipment. The downside of display technologies is their reliance on the natural transcription and translation machinery which limits the accessible chemical space to peptides and biomacromolecules. Considerable progress has been made toward the development of display modalities with a broader chemical space by incorporating unnatural amino acids2 or introducing posttranslational synthetic modification.3,4 Nevertheless, classical small-molecule drug structures remain incompatible with these approaches. A method that could enable the harnessing of the favorable characteristics of small-molecule library screening and display technologies would bear great potential for pharmaceutical © XXXX American Chemical Society
research. DNA-encoded chemical libraries (DECLs) are collections of combinatorial compounds in which each structure is tagged with a DNA identification barcode. DECLs aim to combine the benefits of both chemical library screening and display technologies which would allow the interrogation of small-molecule libraries of unprecedented size rapidly and economically.5 Screening DECLs typically involves affinity selection of libraries against an immobilized protein target similar to phage display, and therefore screening is rapid and inexpensive. Simultaneously, the synthetic methods used to prepare DECLs enable encoding of diverse sets of compounds with potentially druglike characteristics. Lerner and Brenner first suggested DECLs in a theoretical publication in 1992.6 Diverse approaches for synthesizing, screening, and applying DECLs have emerged since then. Early designs combined DNA-encoding with one-bead−one-compound libraries.7 Later DECLs consisted of soluble collections of DNA−small molecule conjugates. Different methods for the generation of DECLs have been proposed including DNAtemplated synthesis,8−10 DNA-encoded routing,11−13 selfassembly induced reactions,14,15 and hybridization-based assembly.16,17 A split-and-pool strategy involving iterative chemical synthesis and DNA encoding steps is currently the most widely used method for DECL preparation.18,19 Furthermore, several encoding methods are available including enzymatic double-strand ligation,19 polymerase extension,18 encoding on beads,7,20 and template-mediated chemical Received: December 4, 2015
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Figure 1. Representative set of library topologies illustrating the combinatorial nature of DNA-encoded chemical libraries (nBB: number of diversity points in library).
ligation.21,22 Libraries encoded with peptide nucleic acids (PNA) are also used for hit discovery (DECL is used throughout this article for all encoded libraries including those containing PNA).17,23−27 The conventional method for DECL screening is incubation of the library in the presence of immobilized target proteins followed by washing steps to remove DNA conjugates of molecules with low target affinity. Sequence enrichment is then analyzed by high-throughput DNA sequencing or using DNA microarray chips. Alternative screening modalities have been proposed including immunoprecipitation,11 chemical cross-linking,28 SDS−PAGE,29,30 and dynamic combinatorial library approaches.31,32 With progressing advancements in DECL technology the research focus has shifted from methodology development to applications in drug discovery. As a result, a growing number of hits for pharmaceutically relevant targets have been appearing in scientific literature. With the emergence of screening hits, new challenges related to the identified structural features are becoming evident.5 This review focuses on the design of DECLs and the properties of screening hits reported in peer-reviewed literature. We analyzed the physicochemical properties of hits using simple parameters that are statistically associated with peroral absorption including Lipinski’s rule of 533 (Ro5; molecular weight MW < 500 Da; calculated octanol/water partition coefficient ClogP < 5; number of hydrogen bond acceptors HA ≤ 10; number of hydrogen bond donors HD ≤ 5) and Veber descriptors (polar surface area PSA < 140 Å2; number of rotatable bonds RotB ≤ 10).34 We further present recently reported hit structures and discuss emerging trends and possibilities associated with this technology. Several excellent reviews are available for readers interested in the technological aspects of library preparation and screening.5,27,35−40
combining several fragments into a single structure often resulted in unacceptably large molecules and such designs had a tendency to yield overly hydrophobic or floppy molecules. Furthermore, in some cases insufficient quality control during library synthesis resulted in impurities that complicated library analysis.41,42 Low scaffold diversity and overreliance on a small set of reactions for library synthesis may also be a reason for the low productivity of combinatorial libraries. Implementing the conclusions derived from combinatorial chemistry will likely be essential for the success of DECLs in drug discovery. Indeed, early efforts in DECL development focused on technological challenges and aimed to demonstrate the principle of encoded library screening. In these studies the molecular properties of hit compounds were of secondary priority, and as a result, most hits from early libraries exhibited structural liabilities and were unattractive for hit-to-lead development. Only in recent years has there been an increased attention with regard to the chemical space of encoded compounds. Library design directly influences the chemical properties of encoded compounds. Important DECL design parameters are the number of diversity points (i.e., sets of building blocks and synthetic cycles) and the assembly geometry (Figure 1). Moreover, the chemical reactions used to connect the diversity points and the building blocks themselves strongly influence the properties of the molecules. Possibly the most fundamental DECL design criterion is the number of diversity points (nBB). This number has an immediate effect on the chemical properties of the encoded compounds. Adding sets of building blocks inevitably increases the molecular mass. In an illustrative example, building blocks have an average MW of 200 Da. In this example, compounds in a library with two diversity points (2BB library) have an average molecular mass of 400 Da (ignoring linking scaffolds), and the MW increases to 600 and 800 Da for 3BB and 4BB libraries, respectively. This trend exemplifies the challenge of retaining druglikeness with increasing number of diversity points. Even for a 3BB library, it is difficult to ensure that the majority of encoded compounds have a molecular weight of 500 Da (Figure 4a). The median MW of DECL hits noticeably increased with the number of diversity points (2BB, 479 Da; 3BB, 499 Da; 4BB, 652 Da; only unoptimized hits) and so did the fraction of hits with MW < 500 Da (2BB, 20 out of 37; 3BB, 14 out of 37; 4BB, 0 out of 24). This tendency is likely a consequence of the increasing average MW of encoded compounds as a result of assembling several building blocks. Interestingly, the MW of some 3BB screening hits was comparable to those of hits isolated from 2BB libraries. Especially, InhA inhibitors identified from a library with a branched design consisting of two sets of terminal building blocks assembled on variable linkers as the third building block (i.e., library L) tended to be favorable in terms of MW.48 Such designs may be an effective way to enhance the structural diversity of libraries without drastically increasing MW of encoded compounds. However, hits from libraries with related overall topologies were less favorable (i.e., libraries H, N, R),23,55,62 highlighting the need to control the size of scaffolds and building blocks. One example where large scaffold elements lead to elevated MW is a library with a Diels−Alder generated 2BB library (DECL D).54,73 In contrast to MW, the ClogP values of a large fraction of hits for 2BB and 3BB hits were in a range that is generally attributed to be druglike (−1 < ClogP < 5; Figure 4b). Problematic ClogP values generally resulted from overly hydrophobic building blocks. Especially early examples of DECLs contained problematically lipophilic fragments.18,54 Applying appropriate filters such as the “rule of 3”74 criteria of lipophilicity (ClogP < 3) to building block selection can straightforwardly help to minimize hydrophobicity issues.59 OffDNA 4BB screening hits mostly segregate into two classes of compounds with either ClogP > 5 or ClogP < 0. Several hits
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DNA-ENCODED LIBRARY SCREENING HITS We analyzed published DECL screening hits to assess the impact of different library design parameters on physicochemical properties (MW, ClogP, HA, HD, PSA, RotB). We excluded compounds optimized from known lead structures (e.g., sulfonamides for carbonic anhydrases) as well as binders of streptavidin71,72 and serum albumins52,59 from this analysis. Of special interest was the influence of the number of diversity points on structural properties of hits. Molecules included in this analysis are summarized in the Supporting Information (Table S1). Information about the screened libraries and the level of structural optimization was lacking for some compounds which complicated our analysis of the chemical space of DECLs. All chemical parameters were calculated using OSIRIS DataWarrior (http://www.openmolecules.org/ datawarrior/). The data set contained the chemical properties of 155 compounds identified in 32 selections of which 111 were direct library hits and 44 were either truncated (fragments at one diversity point were omitted) or medicinal chemistry optimized. For this analysis, we define “hit” as a structure identified directly from a DECL screen and “selection” as a set of experiments performed with a unique combination of a particular library and target protein. In a first analysis, we evaluated the size (MW) and hydrophobicity (ClogP) of published DECL screening hits. Figure 3 shows a scatter graph of MW plotted versus ClogP values. Multiple hits were conformant with both Ro5 criteria for size and hydrophobicity33 (MW < 500 Da; ClogP < 5), validating the successful isolation of potentially druglike compounds directly from DECL screening experiments. However, most molecules were located outside this preferred chemical space. All compounds with favorable characteristics have been reported recently (the color of the markers in Figure 3 indicates the publication year), which reflects efforts to construct libraries with druglike compounds. The Ro5 druglike E
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Figure 4. Distribution of selected physiochemical properties for libraries with different numbers of diversity points. Compounds are coded by color and shape for their degree of optimization (red rhombus, direct screening hit; yellow circle, screening hit truncated at one position; blue triangle, optimized hit compounds). N/S indicates compounds for which no information on the screened library was provided.
whereas others were beyond these values (Figure 4c and Figure 4d). The fraction of hits within the accepted range for these parameters decreased from 2BB to 4BB DECLs. With few exceptions, analyzed compounds complied with the criteria of HD ≤ 5 (Figure 4e).
from 4BB libraries with a triazine core were rather hydrophobic (i.e., DELCs F and J),19,49,70 and it remains to be seen whether this is an intrinsic characteristic of these libraries. In contrast macrocyclic 4BB hit structures based on amide-bond chemistry were rather hydrophilic. Interestingly, optimized leads originating from 4BB libraries often had ClogP values in the preferred range. Excluding building blocks because of lipophilicity considerations may be disadvantageous for protein targets with hydrophobic binding pockets. For example, selections against human serum albumin (HSA)52,59 and heat shock protein 70 (HSP70)23 appeared to be biased to hydrophobic building blocks. Numerous hit compounds had RotB > 10 (Figure 4f). Structural floppiness may originate on the level of both building blocks and scaffolding elements. For illustration, a lysine scaffold adds seven rotatable bonds rendering it challenging to meet the Veber criteria of RotB ≤ 10. Special cases are molecules identified from screening dual-pharmacophore libraries.16,17 In such DECLs, the different fragments are flexibly spaced on a duplex which allows the sampling of distant protein binding pockets. In addition to the challenge of finding suitable connectors the resulting linkers can be lengthy and add a large number of RotB. A good illustration of this problem is an α-1-acid glycoprotein (AGP) binder with 28 rotatable bonds identified from a 2BB dual-pharmacophore library.63 For PSA and HA a portion of hit compounds was in agreement with proposed threshold criteria (PSA < 140 Å2 and HA ≤ 10),
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HIT OPTIMIZATION Physicochemical properties can change significantly during hit optimization, and the characteristics of initial hit compounds are poor predictors of final lead structures. Hits identified from different sources (e.g., HTS, fragment based screening, virtual screening, and natural product sources) tend to change differently during hit optimization.75 We were interested in how chemical properties of DECL screening hits varied during medicinal chemistry optimization (level of optimization is indicated as the shape of data points in Figure 3). Different patterns of property changes during optimization have been observed. The MW of all nonmacrocyclic 3BB and 4BB DECL hits decreased during hit optimization. In several examples, fragments at one diversity point could be removed without significant loss of affinity resulting in a reduction of MW. This situation is illustrated for a pan-inhibitor of sirtuin (SIRT) enzymes identified by screening a 3BB DECL.58 Inhibitory activity toward SIRTs was retained upon replacement of the fragments present at diversity point 1 by a mesylate group; however, the MW decreased significantly (approximately 90 Da). An interesting example of a case where one diversity F
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hydrogen atoms in hits from these libraries. In this analysis, 2BB library hits mostly had lower LEs because of lower potency. For the same targets, direct comparisons between similar 2BB and 3BB library screening results are currently lacking and it remains to be seen whether these trends will be consistent in the future. In conclusion, screening DECLs can provide highly potent molecules with LEs comparable to compounds identified by other discovery methods.
element was unimportant for protein affinity was an inhibitor of p38 mitogen-activated protein kinase (p38 MAPK) identified from a 4BB library (DECL E).19 It was found that triazine derivatives with ethoxy or hydroxy groups at one diversity point were better inhibitors than hit compounds with amines at this diversity point as anticipated in the library design. Therefore, it should be possible to identify druglike lead compounds even from seemingly unfavorable libraries. It is however also evident that for multidiversity point libraries often only a portion of the chemical space of DECLs is actually sampled. For macrocyclic compounds MW remained constant or increased during lead development possibly because removing building blocks would have altered the overall structure unfavorably.76,77 A special case is a binder of the baculoviral IAP repeat X-linked inhibitor of apoptosis (XIAP-Bir3) which was found to be significantly more potent as a dimer and consequently increased drastically in MW during medicinal chemistry optimization.45 The MW of two hits identified from a branched 2BB library with benzimidazole scaffolds increased during lead development.51 In summary, it appears that MW decreases frequently for nonmacrocylic 4BB library hits whereas for 2BB library hits MW increases or remains constant. However, more data are needed to draw general conclusions. Medicinal chemistry optimization also noticeably improved lipophilicities of hit compounds (e.g., structures 13 and 14 in Chart 4).
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DNA-ENCODED LIBRARY SCREENING HIT COMPOUNDS Numerous ligands have been identified in recent years from DECL screening campaigns for a diverse set of proteins, and an increasing fraction of these compounds are conformant with Ro5 and Veber descriptors. Representative examples are summarized in the following paragraphs grouped according to the family of target proteins. Kinases. Screening DECLs yielded several inhibitors of kinases (Chart 1). In 2009, Clark et al. used trisubstituted triazine libraries (DECLs E and F) to identify nanomolar inhibitors of p38 MAPK and Aurora A kinase (structures 1 and 2 in Chart 1), providing early examples of the potential of this technology.19 A convincing example of how DECL screening can provide potent inhibitors with druglike physicochemical properties is a brain-permeable inhibitor (structure 4 in Chart 1) of glycogen synthase kinase 3 (GSK-3).79 Optimization of the initial hit provided inhibitors of GSK-3 with low nanomolar IC50 values and favorable MW and ClogP values. Details concerning the screened library were not provided. A team from GSK recently disclosed inhibitors of phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3Ka) identified from a 3BB DECL (library R) with IC50 values in the single-digit nanomolar range.62 Fragments introduced during synthesis cycle 1 could be omitted without significant loss of binding affinity, yielding inhibitors with good LE (e.g., structure 3 in Chart 1; IC50 = 10 nM; LE = 0.3) without extensive optimization. A potent DECL derived inhibitor of receptorinteracting protein kinase 3 (RIP3) was also reported (structure 5; IC50 = 0.3 nM).80 Kleiner et al. isolated highly specific macrocyclic bisubstrate inhibitors of Src kinase from a DECL; further optimization provided potent Src inhibitors (e.g., structure 6 in Chart 1), some of which exhibited cellular activity at high concentrations.10,76 A notable feature of the mentioned kinase inhibitors is the presence of novel chemotypes. For instance, an unusual binding mode was found for the GSK-3 inhibitor 4, and the macrocycles identified for Src kinase such as 6 are atypical for kinase inhibitors. These examples illustrate how structurally unbiased DECLs may offer value to medicinal chemists by yielding alternative chemotypes for lead development complementing focused HTS libraries for this class of enzymes. Potentially such inhibitors will have different resistance and selectivity profiles than inhibitors derived from alternative hitidentification methods. The identification of novel chemotypes is an often observed feature of DECL hits also for other target classes. Phosphatases. DECLs also provided hits against phosphate ester-hydrolyzing enzymes (Chart 2). A hit with a benzimidazole-indole core was identified for phosphodiesterase 12 (PDE12; identified from DECL V) which could be further improved to subnanomolar potencies in an enzyme inhibition assay (structure 7 in Chart 2; IC50 = 0.8 nM), although optimization was associated with a significant increase of
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LIGAND EFFICIENCY One of the decisive properties of a hit compound is its activity. The diverse nature of the target proteins complicated a meaningful comparative analysis of screening hits. It is however noteworthy that several molecules identified using this technology have single-digit nanomolar or picomolar IC50 values in biochemical assays. Representative examples include inhibitors of phosphodiesterase 12 (PDE12; IC50 = 0.79 nM),51 HSP-70 (best binder IC50 = 0.38 nM),23 and soluble epoxide hydrolase (sEH; IC50 = 0.028 nM and IC50 = 2 nM).22,78 Analysis of ligand efficiencies (LE) revealed several compounds with LE of >0.3 (Figure 5). Leads improved by medicinal
Figure 5. Ligand efficiencies for compounds derived from libraries with different number of diversity points. Compounds are coded by color and shape for their degree of optimization (red, rhombus, direct screening hit; yellow circle, screening hit truncated at one position; blue triangle, optimized hit compounds). N/S indicates compounds for which no information on the screened library was provided.
chemistry optimization exhibited an evident tendency to higher LEs. This result may have been due to enhanced potency or reduced size. However, even some direct screening hits had LE values of >0.3. For reported hits LEs tend to be highest for compounds derived from 3BB libraries. LEs dropped for 4BB and 5BB libraries as a result of the increasing number of nonG
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Chart 1. Representative Kinase Inhibitors Identified by Screening DNA-Encoded Chemical Librariesa
a
Compounds 3−6 were optimized hits; structure 2 is truncated and compound 1 is a direct library screening hit.
DECL hit yielded an orally active compound (GSK2830371;81 structure 8 in Chart 2) with antilymphoma activity in tumor xenografts.81 In a very recent publication, Barluenga et al. reported an inhibitor of protein tyrosine phosphatase 1B (PTP1B) from screening a PNA encoded chemical library in which one set of building blocks consisted of a set of putative phosphotyrosine mimetics (structures of inhibitor and library not shown).82 This discovery adds further support to the assertion that proper selection of building block is essential for screening success. NAD+-Binding Proteins. Several classes of enzymes use NAD+ as a cofactor, and DECL screening has provided inhibitors for such proteins (Chart 3). Researchers at GSK described potent pan-inhibitors for SIRT1/2/3 from screening a 3BB library (DECL K).58 The diacids at diversity point 1 contributed little to binding and could be omitted which thus enhanced the physicochemical properties of these structures. Truncated hits (e.g., structure 9 in Chart 3) had single-digit nanomolar IC50 values (SIRT1 = 4 nM; SIRT2 = 1 nM; SIRT3 = 7 nM) in a biochemical assay and high LE values (e.g., 0.49 for SIRT2). Screening of two 2BB DECLs with branched designs (DECLs O and P) yielded screening hits with a novel dihydrouracil fragment from both libraries combined with varied aromatic groups at the second position.59,61 Resynthesis of these hits (e.g., structures 10 and 11 in Chart 3) afforded
Chart 2. Representative Phosphatase Inhibitors Identified by Screening DNA-Encoded Chemical Librariesa
a Both compounds resulted from efforts to optimize properties of the primary hits.
MW.51 An allosteric inhibitor of wild-type p53-induced phosphatase (Wip1) based on a capped peptide structure was identified from a DECL screen.81 This study is one of the rare examples where DECL screening hits were directly compared to HTS derived compounds for the same target. In this particular study, the structures identified from both techniques exhibited obvious structural similarities. Optimization of the
Chart 3. Representative Inhibitors of NAD+-Binding Proteins Identified by Screening DNA-Encoded Chemical Librariesa
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Compounds 9 and 12 were obtained from efforts to optimize the properties of primary hits; compounds 10 and 11 are primary library screening hits. H
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inhibitors of tankyrase 1 (TNKS1) with nanomolar IC50 values in an enzyme inhibition assay (10, 290 nM; 11, 250 nM). Selectivity relative to TNKS2 was observed for some but not all structures, and inhibition was comparable to or stronger for poly (ADP-ribose) polymerase 1 (PARP1). For instance, 10 inhibited PARP1 with an IC50 value of 40 nM before any optimization, providing an unreported chemotype for this well studied drug target. An inhibitor of the enoyl ACP-reductase InhA from Mycobacterium tubercolosis was found from screening the 3BB library L.48 Analysis of the crystal structure revealed that the inhibitor bound to the enzyme active site without displacing NADH. The optimized hit (structure 12 in Chart 3) had potent in vitro (IC50 = 4 nM) activity but was inactive in an acute tuberculosis infection model.48 Proteases. Inhibitors derived from DECL screening were identified for several proteases (Chart 4). Examples included
Using benzamidine as a lead fragments, Neri and co-workers discovered potent and selective inhibitors of several serine proteases using single- and dual-pharmacophore libraries (structures not shown).83,84 Other examples of using DECL screening for hit optimization include reports of affinityoptimized carbonic anhydrase IX (CAIX) binders.63 Covalent inhibitors of cysteine proteases were also identified from screening PNA-encoded chemical libraries.29,30 Soluble Epoxide Hydrolase. Two groups reported potent inhibitors of soluble epoxide hydrolase (sEH; Chart 5). Chart 5. Representative Inhibitors of Soluble Epoxide Hydrolase (sEH) Identified by Screening DNA-Encoded Chemical Librariesa
Chart 4. Representative Inhibitors of Proteases Identified by Screening DNA-Encoded Chemical Librariesa
a Structure 16 was a direct DNA-encoded library screening hit and structure 17 obtained by optimization of a primary screening hit.
Researchers at X-Chem reported an inhibitor (structure 16 in Chart 5) identified directly from 3BB library Q with IC50 = 2 nM in an enzyme inhibition assay and druglike structure from a Ro5 and Veber perspective.22 A triazine-based inhibitor was also found for the same target.78 GSK2256294A85 (structure 17 in Chart 5), an optimized structure, inhibited this target very potently in vitro (IC50 = 0.028 nM) and with high ligand efficiency (LE = 0.45).85 This molecule is currently evaluated in a phase I clinical trial (clinical identification number, NCT02006537). Miscellaneous Targets. Screening a 4BB library yielded a potent hit for the hepatitis C virus protein NSB4.50 Optimized hit compounds (GSK0109, 50 [2-(4-(4-chlorophenyl)-5phenyl)pyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-9-yl](phenyl)methanone, structure not shown; GSK4809,50 structure 18 in Chart 6) inhibited viral replication at concentrations below
