A Focused DNA-encoded Chemical Library for the Discovery of

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A Focused DNA-encoded Chemical Library for the Discovery of Inhibitors of NAD+-dependent Enzymes Lik Hang Yuen, Srikanta Dana, Yu Liu, Samuel I Bloom, Ann-Gerd Thorsell, Dario Neri, Anthony J. Donato, Dmitri B. Kireev, Herwig Schüler, and Raphael M. Franzini J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08039 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 11, 2019

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Journal of the American Chemical Society

A Focused DNA-encoded Chemical Library for the Discovery of Inhibitors of NAD+-dependent Enzymes Lik Hang Yuen,†,‡ Srikanta Dana,†,‡ Yu Liu,‖ Samuel I. Bloom,‖ Ann-Gerd Thorsell,§ Dario Neri,ǂ Anthony J. Donato,‖ Dmitri Kireev,× Herwig Schüler,§ Raphael M. Franzini†,* † Department

of Medicinal Chemistry, University of Utah, 30 S 2000 E, Salt Lake City, UT-84112, United States Department of Biosciences and Nutrition, Karolinska Institutet, Hälsovägen 7c, 14157 Huddinge, Sweden ǂ Department of Pharmaceutical Sciences, ETH Zürich, Vladimir Prelog Weg 3, 8093 Zürich, Switzerland × Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC-27599, United States ‖ Department of Internal Medicine, University of Utah, 500 Foothill Drive, Salt Lake City, UT-84148, United States §

ABSTRACT: DNA-encoded chemical libraries are increasingly used in pharmaceutical research because they enable the rapid discovery of synthetic protein ligands. Here we explored whether target-class focused DNA-encoded chemical libraries can be cost-effective tools to achieve robust screening productivity for a series of proteins. The study revealed that a DNA-encoded library designed for NAD+-binding pockets (NADEL) effectively sampled the chemical binder space of enzymes with ADP-ribosyltransferase activity. The extracted information directed the synthesis of inhibitors for several enzymes including PARP15 and SIRT6. The high dissimilarity of NADEL screening fingerprints for different proteins translated into inhibitors that showed selectivity for their target. The discovery of patterns of enriched structures for six out of eight tested proteins is remarkable for a library of 58,302 DNAtagged structures and illustrates the prospect of focused DNAencoded libraries as economic alternatives to large library platforms.

Introduction A fundamental challenge in pharmaceutical research is the identification of molecules that tightly bind to targets of interest; economic, fast, and reliable hit discovery technologies are critical for the development of new medicines. One approach that is increasingly used to discover drug hits is to screen DNA-encoded chemical libraries (DECLs),1 combinatorial libraries in which the structure of each molecule is encoded by conjugated DNA identifier sequences.2 DNAtagging makes it possible to discover hits by simple affinity selection protocols, which include incubation of libraries with targets, elution of unbound conjugates, and amplification of retained DNA tags for DNA sequencing (Fig. 1A).3 This workflow enables scientists to rapidly and inexpensively screen small-molecule libraries of vast sizes, and it has resulted in numerous ligands for diverse proteins.4

Discovering hits in few hours, at low screening cost, requiring microgram of proteins, using standard instrumentation, and obviating the development of screening assays should in principle promote drug development efforts independent of sophisticated screening centers.5 However, most reported hits were identified from platforms of DECLs with millions to billions of encoded molecules.4b, 4h, 6 The high cost and labor of constructing such library collections constitutes the principal obstacle to the widespread use of this technology. Additionally, lacking sequencing depth7 and library homogeneity8 can impede effective hit triaging for ultra-large libraries and result in tedious hit validation efforts. Accordingly, there is a need for alternative design concepts to access the transformative potential of DECLs. The pursuit of DECLs designed for specific classes of target proteins is a promising approach to popularize this technology. The established effectiveness of focused conventional libraries make them ubiquitous in pharmaceutical research.9 However, the performance of corresponding DECLs remains surprisingly unexplored even though they might provide robust screening productivity at a fraction of the costs of large one-fit-all platforms. In one report, Barluenga et al. discovered inhibitors of PTP1B and TCPTP from a library designed to target tyrosine phosphatases.10 Another study used a library of DNA-encoded tripeptides with benzamidine at the P1 position to find potent trypsin inhibitors.11 Further efforts are needed to determine the generality of this concept and to learn what degree of screening success is possible for structurally focused DECLs. Here we report the first DECL designed to target NAD+-binding pockets, and we study its utility to sample the chemical binder space of NAD+-dependent enzymes. This NAD+-mimicking DNA-encoded chemical library (NADEL; Fig. 1) contains 58,302 compounds consisting of combinations of 527 fragments arranged in a branched geometry. Screening NADEL provided structurally novel inhibitors for several enzymes with ADP-ribosyltransferase activity despite the library’s

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straightforward design and comparatively small size. For example, unoptimized hits of PARP15 and SIRT6 were among the most potent small-molecule inhibitors of these proteins. Furthermore, the screening results of NADEL for different isoforms afforded inhibitors that were selective to their

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respective targets. The demonstration that a small target-class focused DNA-encoded library affords consistent screening productivity for several members of a protein family should encourage resource-effective implementations of this technology in medicinal chemistry.

Figure 1. Design of NAD+-mimicking DNA-encoded chemical library (NADEL). A) General workflow used to screen NADEL. B) Structure of NADEL and relationship to NAD+. C) Representative inhibitors of ADP-ribosyltransferases. Green color indicates pharmacophores present in NADEL. D) Examples of fragments at FS-1 targeting nicotinamide-binding pockets. Red color highlights functionalities directed to the carboxamide-binding residues.

Results and Discussion Design and synthesis of NADEL: A DNA-encoded library targeting NAD+-binding pockets. We conceived NADEL to target NAD+-binding pockets of enzymes dependent on this cofactor (Fig. 1) such as ADP-ribosyltransferases, sirtuin family protein deacylases, and oxidoreductases. The design of NADEL adhered to two principles to promote effective discovery of ligands of such enzymes. First, NADEL imitates the overall structure of NAD+ by juxtaposing two fragment sets (FS-1/2) on a 2,3-diaminopropionamide scaffold (Fig 1B). The 2,3-diaminopropionamide scaffold was chosen as the structurally most compact linker to assemble two carboxylic acid fragments. Rather than mimicking the specific interactions of the diphosphate-ribose moiety, it reproduces the overall shape of the cofactor. Many building blocks contain short linkers of variable lengths between the core fragment and the

carboxylic acid group, which position the fragments at different distances and allow sampling different portions of the binding pocket. Second, the 158 fragments at FS-1 (Fig 1D; the complete list of building blocks used for the synthesis of NADEL is available in Tables S1 and S2 and an analysis of scaffold diversity is provided in Figure S1 in the Supporting Information) contain hydrogen donor/acceptor patterns reminiscent of the carboxamide group of NAD+ and similar to the ones that are often present in inhibitors of such proteins (Fig. 1C).12 The combination with 369 structurally unbiased fragments at FS-2 endows NADEL with a total of 58,302 DNAtagged compounds. The preparation of NADEL followed a reported synthesis protocol (see Supporting Information for details).4j, 13 Validation experiments with streptavidin as a target provided the expected enrichment of desthiobiotin-containing compounds (Fig. S4 in the Supporting Information) supporting the correct assembly of the library.


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Figure 2. Discovery of inhibitors for PARP1 by screening NADEL. A) Three-dimensional high-throughput DNA sequencing results of NADEL screen for PARP1. Each marker corresponds to a compound in the library whose structure is defined by the combination of fragments at FS-1/2 specified on the x/y-axes; z-values indicate normalized sequence counts (NSC). Sequence enrichment is highlighted by color and marker size; compounds with NSC values 10). Structures of representative fragments found in hit compounds are shown. C) A docking model of A65 (green) binding to PARP1 (magenta; 6BHV); the docking pose and an interaction diagram for A65-(CONH2)-B101 are shown in Fig. S11a in the Supporting Information. D) Structures of synthesized derivatives of representative PARP1 screening hits and inhibitory potencies determined in an enzyme activity assay. Synthesized compounds included structures with and without the carboxamide used for DNA-attachment in NADEL because the influence of the linker is unknown. [a] IC50 values for A65-containing compounds are provided in Table 1.

Sampling the chemical binder space of PARP1 with NADEL. As the first step to assess the performance of NADEL, we tested whether it can discover hit compounds for poly(ADPribose) polymerase 1 (PARP1). PARP1 polymerizes ADPribose chains to regulate DNA-repair and damage response,14 and it is a drug target for ovarian and breast cancer patients with BRCA germline mutations.15 Four PARP1 inhibitors have received regulatory approval by 2019 (olaparib,15b rucaparib,16 niraparib17, and talazoparib18), and the medicinal chemistry of PARP1 is well studied.19 We screened NADEL for enzymatically biotinylated (Avi-tag) human PARP1 immobilized on magnetic beads (for details on protein

constructs see Table S3 in the Supporting Information) followed by the identification of enriched compounds by PCR amplification and sequencing of DNA tags as previously reported (Fig. 1A; see Supporting Information for details).3b PARP1 screening experiments resulted in elevated normalized sequence counts (NSC; formula S1 in the Supporting Information) for numerous NADEL compounds, which contrasts the homogeneous sequence distribution of the initial library (Figs. 2A,B and S3 in the Supporting Information). A series of compounds containing the 3,5,6,7-tetrahydro-4Hcyclopenta[4,5]thieno[2,3-d]pyrimidin-4-one pharmacophore A65 with NSC values of >100 dominated the sequencing



fingerprint (Fig. 2A,B). To validate hits, we synthesized compounds with the two fragments assembled on a 2,3diaminopropionamide scaffold (Fig. 2D; all synthetic procedures are provided in the Supporting Information). Analysis of such derivatives of the two most highly enriched A65-hits containing 4-imidazoleacetic acid (B101) and 1-methyl-3-indoleacetic acid (B123) confirmed potent inhibition of PARP1 in a histone parylation assay (BPS bioscience) with IC50 values of 180 nM for A65-(CONH2)B101 (Nomenclature: compounds are named according to pharmacophores at FS-1 and FS-2 with the substituents at the ethylene group in parenthesis between the two) and 190 nM for A65-(CONH2)-B123 (Table 1 and Table S6 in the Supporting Information). To test whether the linker is involved in protein binding, we synthesized the N-methylated compound A65(CONHMe)-B101, and tests revealed that the alkyl group has little influence on target affinity (IC50 = 170 nM; Table 1). Table 1. Inhibition of PARP1 and PARP2 by structures containing the fragment A65 PARP1 IC50 (pIC50)

PARP2 IC50 (pIC50)

A65-(CONH2)-B101

180 nM (6.74 ± 0.28)

21 nM (7.67 ± 0.04)

A65-(CONH2)-B123

190 nM (6.71 ± 0.46)

nd.

A65-(CONHMe)-B101

170 nM (6.77 ± 0.02)

nd.

A65

82 nM (7.09 ± 0.015)

35 nM (7.46 ± 0.028)

A65-(CONH2)-NH2[a]

71% (100 nM)

70% (100 nM)

[a] Single

concentration measurement.

Consistently high enrichment values for a large fraction of structures of a fragment, as observed for A65, often indicate that this fragment is predominantly responsible for binding affinity of the compounds.2b In such cases, it is possible to remove part of the molecules without decreasing the binding affinity. We therefore tested whether A65 alone was an inhibitor of PAPR1, and A65 inhibited the parylation activity of PARP1 with an IC50 value of 82 nM. Interestingly, closely related structures lacking the carboxylic acid functionality were reported to have IC50 values of 12-27 μM.20 A65 with the linker but without the B101 fragment also inhibited PARP1 (71% inhibition at 100 nM). We further tested whether the A65 derivatives were also inhibitors of the structurally and functionally closely related isoform PARP2.21 Both A65-(CONH2)-B101 and A65 potently inhibited PARP2 with IC50 values of 21 nM and 35 nM,

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respectively. The compounds were stable for >72 h in serum (Fig. S12 in the Supporting Information). This outcome confirms that screening NADEL can be a source for potent pharmacophores for NAD+-binding proteins. To analyze the binding mode of A65, we performed docking studies. According to the model, the heterocycle of A65 forms hydrogen bonds with Gly863 and stacks to Tyr907 (Fig. 2C). These interactions are typical for PARP1 ligands. In the model, the carboxylic acid group forms hydrogen bonds with Ser864 and Asn868 (Fig. 2C). In case of A65-(CONH2)-B101, the linker and the B101 fragment points outward into the NAD+pocket forming hydrogen bonds with the backbone amide of Arg878 and Gln875 (Figs. S13 and S14 in the Supporting Information). Other NADEL hits for PARP1 were structurally diverse (Fig. 2B). Fragments at FS-1 included quinazolinones (A44, A115), 1-phenyl-5,6-dihydrouracil (A11), quinazoline-1,2(3H,4H)dione (A108), benzamide (A45), and pyrazole-3-sulfonamide (A23). Complementing fragments at FS-2 included 3-aryl1,2,4-oxadiazoles (B190, B259) and imidazoles (B101, B295). Derivatives of representative hits with different linkers were synthesized, tested and were found to inhibit PARP1 with IC50 values in the low micromolar range (Fig. 2D; A23-(H)-B184: IC50 = 1.9 µM; A153-(CONH2)-B356: IC50 = 1.4 µM; A11-(H)B101: IC50 = 1.4 µM; A108-(H)-B101: IC50 = 1.5 µM). These molecules overall were consistent with the established medicinal chemistry of PARP112b, 19 and concomitantly revealed new structural patterns. For example, A153 resembles the 1(2H)-phthalazinone core of olaparib (Fig. 1C) while its combination with a 1-indanone such as B356 is unprecedented. A23-(H)-B184 is an example of a hit that is chemically distinct from typical PARP1 inhibitors. These results establish that screening NADEL can reveal information on the chemical binder space of this therapeutically important NAD+-binding protein. Discovery of inhibitors of mono-ADP-ribosyltransferases. To explore the general utility of NADEL for discovering hits to members of the PARP protein family, we next performed affinity selections for the mono-ADP-ribosyltransferases PARP4, PARP10, PARP12, PARP14, PARP15, and PARP16 (for details on constructs and protein expression protocols see Tables S4 in the Supporting Information). Only a limited number of studies have explored the medicinal chemistry of these enzymes,22 and chemical probes to elucidate their important roles in cellular processes23 are typically unavailable. Screening NADEL provided structures with highly enriched sequence counts for four of these targets: PARP10, PARP12, PARP14, and PARP15 (Fig. 3A; results for PARP4 and PARP16 are shown in Fig. S5 in the Supporting Information).


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Figure 3. Library screening results for the mono-ADP-ribosyltransferases PARP10, PARP12, PARP14, and PARP15. A) Three-dimensional scatter plots of NADEL screening results (cutoff: normalized sequence count (NSC) >20). B-D) Top-views of screening results with structures of selected fragments present in compounds with high sequence enrichment. Shown structures are hit compounds tested in biochemical assays with IC50 values provided. Top view for PARP14 containing structures of selected fragments is shown in Fig. S6 in the Supporting Information. E) Docking model of A82-(CONHMe)-B354 (green) to PARP10 (magenta; 5LX6). F) Docking model of A101(CONH2)-B322 (green) to PARP15 (magenta, 4F09). Interaction maps are shown in Fig. S11 in the Supporting Information.



We synthesized and tested small-molecule derivatives of representative hits for PARP10, PARP12, and PARP15. For PARP14, structural features associated with elevated NSC values (Fig. 3A and S6 in the Supporting Information) were consistent with known inhibitors of this target. Discovered fragments included structures closely related to the metasubstituted carboxamide pharmacophores present in previously reported inhibitors of PARP14 (e.g. 3 in Fig. 1C),22c, 22d, 24 and a derivative resembling a thieno[2,3-d]pyrimidine-4-one fragment present in a disclosed PARP14 inhibitor.24b Therefore, NADEL unraveled structural features relevant to inhibitors of this target, and we did not pursue PARP14 hits further because several potent inhibitors are available for this target.22c, 22d, 24 The results for PARP10, PARP12, and PARP15 are summarized below. PARP10: The mono-ADP-ribosyltransferase PARP10 is involved in immune signaling,25 control of metabolism,26 response to DNA damage,27 and transcriptional regulation.28 Venkannagari et al. discovered 4-(4-oxomethylphenyloxy)phenylcarboxamide as a privileged structure for PARP10,22e which has been incorporated into several inhibitors of this enzyme (see 5 in Fig. 1C).22a, 22b In agreement, this fragment (A47; Fig. 3B) emerged among the screening hits, providing a validation for NADEL’s ability to report on the fragment binder space of mono-ADP-ribosyltransferases. The most enriched compounds however contained an unreported 6-carboxy tetralone fragment (B354) at FS-2. The two molecules that were evaluated (Fig. 3B), A82-(CONHMe)-B354 and A34-(CONHMe)-B354, inhibited PARP10 with IC50 = 6.0 μM and 24.6 μM, respectively. Interestingly, the docking pose for the first compound (Figs. 3E and S14B in the Supporting Information) is reminiscent of a binding mode identified recently,22a, 22b where the B354 fragment targets a sub-pocket adjacent to the active site loop, whereas traditional PARP inhibitors extend into the adenine sub-pocket. Therefore, further development of these molecules presents an opportunity to arrive at selective inhibitors of PARP10. PARP12: The medicinal chemistry of PARP12 remains largely unexplored despite reports that this enzyme has important biological functions.29 A68/B259 appeared as the most highly enriched fragment pair in the NADEL screening data for PARP12 (Fig. 3C), combining a para-substituted benzamide (A68) with a 3-(4-pyridinyl)-1,2,4-oxazole (B259). We measured an IC50 value of 38.0 μM for A68-(CONH2)-B259 in an auto-ADP-ribosylation assay. The propylcarboxamide derivative was a slightly more potent PARP12 inhibitor (IC50 = 33.5 μM), whereas removal of the carboxamide significantly reduced activity (~60 % inhibition at 100 uM). A68-(CONH2)B259 is a promising starting point for the development of the first potent probes for this enzyme, which is upregulated in many cancers. PARP15: PARP15 is a macrodomain-containing mono-ADPribosyltransferase expressed at high levels in B-cell lymphoma.30 Of the numerous NADEL screening hits, a series of molecules containing the phthalhydrazide fragment A101 had the highest NSC values (Fig. 3D). Synthesis of the two most enriched structures and evaluation in an enzymatic activity assay showed that A101-(CONH2)-B322 containing a m-toluic acid fragment inhibited PARP15 with an IC50 of 200 nM, and

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the inhibitory potency of A101-(CONH2)-B114 was 970 nM. We further tested the methylcarboxamide derivative A101(CONHMe)-B322 and found that it had an IC50 value of 510 nM. This result agrees with previous observations that the attachment linkers can affect binding affinity in unpredictable ways.4j, 31 A101-(CONH2)-B322 showed no signs of decomposition in 72 h (Fig. S12 in the Supporting Information). A docking analysis of A101-(CONH2)-B322 found the phthalhydrazide structure to occupy the nicotinamide pocket of PARP15 forming hydrogen bonds with several backbone residues (Figs. 3F and S14C in the Supporting Information). The m-toluic interacted with Lys559 by a hydrogen bond and a cation-π interaction. To our knowledge, A101-(CONH2)-B322 is the most potent inhibitor reported for PARP15, and the identified phthalhydrazide fragment will be valuable for the future development of chemical probes for this target. Identification of an inhibitor of sirtuin 6 (SIRT6). Having established the utility of NADEL for the discovery of inhibitors of PARPs, it remained to be verified whether the library could also provide hits for other NAD+-dependent enzymes. We selected sirtuin 6 (SIRT6) as a model target for this technology evaluation study because this enzyme has mono-ADPribosyltransferase activity32 in addition to its function as a lysine deacylase.33 SIRT6 exhibits pronounced age-delaying effects,34 and loss of SIRT6 function promotes the onset of neurodegeneration,35 cardiac disfunction,36 and cancer.37 Screening of NADEL against biotinylated recombinant SIRT6 (Fig. S2 in the Supporting Information) returned the 5-aminocarbonyl-uracil fragment A127 combined with the 3pyrinidyl-1,2,4-oxadiazole structure B178 as putative SIRT6 ligands (Fig. 4). We synthesized three 1,2-diaminopropioamide derivatives containing different carboxamides at the linker attachment site (Fig. 4B). A127-(CONHPr)-B178 (IC50 = 6.7 μM) and A127-(CONHMe)-B178 (IC50 = 9.2 μM) inhibited SIRT6 in a commercial demyristoylation assay (BPS bioscience; see Supporting Information for details). Loss of alkyl groups from the carboxamide decreased the inhibitory activity (IC50(A127-(CONH2)-B178) >20 μM). The stability of A127-(CONHPr)-B178 in serum was tested using HPLC and the compound showed no decomposition after 72 h in serum (Fig. S12 in the Supporting Information). In a docking model, A127-(CONHMe)-B178 occupied the cleft in the Rossmannfold binding the NAD+ cofactor (Figs. 4C and S14D in the Supporting Information). The A127 fragment interacted with the C-site that binds nicotinamide stacking to Tyr257 and forming hydrogen bonds to Thr57 and Leu241; B178 reaches out into the B-site, which accommodates the nicotinamide ribose ring.38 The carboxamide group pointed towards the solvent compatible with attachment of the DNA-linker in NADEL. This data would indicate that A127-(CONHR)-B178, in addition to being structurally distinct from reported sirtuin inhibitors, also engages the target in an unconventional binding mode.39 A127-(CONHPr)-B178 is among the most potent SIRT6 inhibitors known except for two catechins (IC50 = 2.5 and 5.4 μM),40 which generally are promiscuous inhibitors of diverse proteins. Reported synthetic SIRT6-specific inhibitors have IC50 values of ≥20 μM despite substantial medicinal chemistry optimization efforts.41 The discovery of a structurally novel SIRT6 blocker demonstrates that NADEL is effective for the identification of inhibitors of proteins of different NAD+dependent enzyme families.


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Journal of the American Chemical Society which is consistent with excessive cellular DNA damage but could also be the result of A127-(CONHPr)-B178 interfering with other cellular processes. The increased formation of 53BP1 foci in HUVECs is intriguing given that SIRT6 accelerates the recruitment of 53BP1 to sites of DNA damage.32b However, the effects of SIRT6 are highly pleiotropic and dependent on the cellular environment.

Figure 4. Discovery of SIRT6 inhibitor from screening NADEL. A) Scatter plot of NADEL sequencing data for SIRT6. B) Structure of tested derivatives of SIRT6 screening hit A127/B178 and IC50 values of inhibition of SIRT6 by derivatives of this hit in a demyristoylation assay. C) Predicted binding interaction of A127(CONHMe)-B178 with SIRT6 based on simulation (interaction map is shown in Fig. S14d in the Supporting Information).

Preliminary evaluation of A127-(CONHPr)-B178 in cells. To test whether A127-(CONHPr)-B178 is membrane permeable and active in a biological context, we performed cellbased activity assays (Fig. 5). SIRT6 activity is associated with DNA repair.34b We therefore analyzed the effect of A127(CONHPr)-B178 on DNA-damage markers in primary Human Umbilical Venous Endothelial Cells (HUVECs). Exposure of HUVECs to 5 μM of A127-(CONHPr)-B178 resulted in elevated levels of p-γ-H2AX by Western blot analysis (1.46-fold increase; p = 0.036; Fig. 5A). Increase in p-γ-H2AX foci was observed for SIRT6 knockdown in HUVECs42 and other epithelial cells.43 We further observed a significantly increased frequency of HUVEC cells with 53BP1 foci (54% of cell treated with compound relative to 4.7% in control cells; p = 0.0012; Fig. 5A and Fig. S16 in the Supporting Information). Previous studies have shown that p-γ-H2AX foci caused by lack of SIRT6 function is associated with telomere damage.42-43 A127(CONHPr)-B178 indeed increased the number of telomeredisfunction induced foci (TIF; Fig. S17 in the Supporting Information), which is in agreement with the role of this enzyme with telomere maintenance,43 and reported results in HUVECs.42 At higher concentrations, A127-(CONHPr)-B178 increasingly induced apoptosis in HUVECs (data not shown),

Figure 5. Phenotypic effects of A127-(CONHPr)-B178 in cells. A) Impact of A127-(CONHPr)-B178 on DNA-double strand breaks in Primary Human Umbilical Venous Endothelial Cells (HUVECs). Left: Quantification of HUVEC cells with 53BP1 foci treated with compound relative to control cells (error bar: standard error). Right: Determination of p-γ-H2AX levels by Western blot analysis. B) Analysis of p21 levels, a downstream marker of cellular senescence (western blot) in HUVECs treated with A127-(CONHPr)-B178 (5 μM) relative to control. C) Induction of premature senescence of HUVEC by treatment with A127-(CONHPr)-B178 (5 μM) relative to control as analyzed by SA-β-gal staining. D) Dose-dependent effect of A127-(CONHPr)-B178 on export of TNF-α from LPSactivated THP-1 cells. (Error bars: standard deviation; p-value determined by two-tailed t-test).

DNA-damage caused by SIRT6 depletion can activate a p53 response and lead to upregulation of p21 and induction of senescence in HUVECs.42 Treatment with A127-(CONHPr)B178 (5 μM) caused 3.8% of cells to become senescent as quantified by SA-β-gal staining relative to 2.2% in controls (1.7-fold increase; p = 0.039; Fig. 5C) and increased p21 levels 1.5-fold (p = 0.0005; Fig. 5B). These results are in agreement



with reported results in these cells.42 The induction of apoptosis prevented measuring the response at higher concentrations of A127-(CONHPr)-B178. To further confirm the SIRT6-inhibiting activity of A127(CONHPr)-B178 in cells, we investigated the compound’s effect on TNF-α export (Fig. 5D). SIRT6 demyristoylates TNF-α and regulates its extracellular release.33c, 44 We treated LPS-stimulated THP-1 monocytes with increasing concentrations of A127-(CONHPr)-B178 and measured the levels of TNF-α in the medium using an ELISA assay (Invitrogen). A dose-dependent decrease in the TNF-α levels was observed, reaching an approximately 40% reduction at 100 μM in agreement with the trend observed for SIRT6 silencing,33c, 44 and other SIRT6 inhibitors.41a The observation that inhibition does not saturate within the measured concentration range could indicate that cell permeability is incomplete or that the intracellular compound distribution is suboptimal. A127-(CONHPr)-B178 showed no concentrationdependent effect on the viability of THP-1 cells (Fig. S18 in the Supporting Information). These phenotypic data provide evidence that A127-(CONHPr)B178 is active in cells. Importantly, A127-(CONHPr)-B178 is an unoptimized hit that was directly discovered from NADEL, and further improvement of the potency and compound properties will be needed to develop this molecule into a chemical probe for biochemical studies.

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NADEL provides target-selective screening hits. Conserved structural features of NAD+-binding pockets make it challenging to develop molecules that selectively inhibit specific PARPs or defined SIRT isoforms.12a, 45 While comparing DECL screening outcomes for several proteins can provide information on target-selectivity of hits,46 this possibility remained untested for enzyme families. In case of NADEL, a primary concern was that screening fingerprints for related PARPs would largely overlap because of the focused library design. However, each protein had distinct patterns of enriched compounds (Fig. 6A). We profiled the selectivity of A65-(CONH2)-B101 (PARP1), A101-(CONH2)-B322 (PARP15), and A82-(CONHMe)-B354 (PARP10) by measuring the inhibition of the screened PARPs PARP1, PARP10, PARP12, PARP14, and PARP15 at an inhibitor concentration of 10 μM. All molecules inhibited the enzyme most potently for which they were discovered (Fig. 6BD). A65-(CONH2)-B101 completely inhibited PARP1 under these conditions, and the mono-ADP-ribosyltransferases retained >85% of their activity. A101-(CONH2)-B322 modestly inhibited PARP10 (31% at 10 μM), and A82-(CONHMe)-B354 showed some off-target inhibition of PARP12 (19.5% at 10 μM). This result confirmed that the relative sequence enrichment data accurately reflected the selectivity of the synthesized hit compounds.

Figure 6. Divergent screening fingerprints enable the discovery of target-selective inhibitors. A) NADEL screening results for selected enzymes with ADP-ribosyltransferase activity (see box; cutoff: NSC >20). Selectivity profiles of screening hits B) A65-(CONH2)-B101 (PARP1 hit), C) A82-(CONHMe)-B354 (PARP10 hit), D) A101-(CONH2)-B322 (PARP15 hit), and e) A127-(CONHPr)-B178 (SIRT6 hit). Darker colors indicate proteins for which NADEL screening was performed, and lighter colors specify PARPs that have not been screened with NADEL. The name of the target protein for which the hit has been discovered is highlighted by color. Values and error bars are provided in Tables S7 and S8 in the Supporting Information).


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We extended our analysis of the selectivity to a broader panel of PARPs including PARP2, PARP3, TNKS1 (PARP5a), PARP6, PARP7, PARP8, and PARP11. As described above, A65-(CONH2)-B101 is a potent inhibitor of PARP2. Interestingly, this molecule also inhibited PARP11 (100% at 10 μM; 20% at 100 nM). Little is known about the chemical inhibitor space of PARP11 and only recently has a nanomolar inhibitor (IC50 = 14 nM) been identified, which shares with A65 a quinazolinone core structure.47 At 10 μM, A65-(CONH2)B101 inhibits PARP3 (66%) and PARP7 (51%), but these inhibitory potencies are significantly lower than those for PARP1, PARP2, and PARP11. In case of A101-(CONH2)B322, some inhibition of PARP11 (41% at 10 μM) was observed in addition to PARP10, whereas for all other PARPs inhibition was 50-fold selective for that target relative to all other tested PARPs. This level of selectivity is striking for an unoptimized hit considering the close structural similarity of the NAD+-binding pocket of PARPs.48 In case of the PARP10 hit A82-(CONHMe)-B354, a 16% inhibition for PARP8 was observed in addition to that of PARP12, and the remaining enzymes were inhibited

A Focused DNA-encoded Chemical Library for the Discovery of

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