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Phenotypic screen for cardiac regeneration identifies molecules with differential activity in human epicardium-derived cells versus cardiac fibroblasts Amalia I. Paunovic, Lauren Drowley, Anneli Nordqvist, Elke Ericson, Elizabeth Mouchet, Anna Jonebring, Gunnar Grönberg, Alexander J. Kvist, Ola Engkvist, Martin R Brown, Karin Gedda, Maria-Jose Goumans, Qing-Dong Wang, and Alleyn T. Plowright ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00683 • Publication Date (Web): 04 Nov 2016 Downloaded from http://pubs.acs.org on November 7, 2016

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ACS Chemical Biology

1 of 18 Phenotypic screen for cardiac regeneration identifies molecules with differential activity in human epicardium-derived cells versus cardiac fibroblasts AUTHOR INFORMATION Amalia I. Paunovic,‡,§ Lauren Drowley,†,§,* Anneli Nordqvist,† Elke Ericson,‡ Elizabeth Mouchet,¥ Anna Jonebring,‡ Gunnar Grönberg,† Alexander J. Kvist,‡ Ola Engkvist,‡ Martin R. Brown,║ Karin Gedda,‡ Marie-José Goumans,⊥ Qing-Dong Wang,† and Alleyn T. Plowright† § these authors contributed equally to this work † Cardiovascular and Metabolic Diseases, ‡ Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Pepparedsleden 1, Mölndal, 431 83, Sweden ¥ Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, Cheshire, UK ║ Discovery Sciences, AstraZeneca R&D Darwin, 310 Milton Science Park, Milton Rd., Cambridge, CB4 0WG, UK ⊥ Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands * Corresponding author: [email protected]

ABSTRACT Activation and proliferation of resident cardiac progenitor cells has therapeutic potential to repair the heart after injury. However, research has been impeded by lack of well-defined and characterized cell sources and difficulties in translation to screening platforms. Here we describe the development, validation, and use of a 384 well phenotypic assay in primary human epicardium-derived cells (EPDCs) to identify

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2 of 18 compounds that induce proliferation while maintaining the progenitor phenotype. Using this assay, we screened 7400 structurally diverse compounds where greater than 90% are biologically annotated and known to modulate a broad range of biological targets. From the primary screen, we identified and validated hits and expanded upon the lead molecules of interest. A counterscreen was developed in human cardiac fibroblasts to filter out compounds with a general proliferative effect, after which the activity of selected molecules was confirmed across multiple EPDC donors. To further examine the mechanism of action of compounds with annotated targets, we performed knockdown experiments to understand whether a single known target was responsible for the proliferative effect, confirming results with protein expression and activity assays. Here we were able to show that the annotated targets of compounds of interest were not responsible for the proliferative effect, which highlights potential differences in cell types and signaling pathways and possible polypharmacology. These studies demonstrate the feasibility of using relevant human primary cells in a phenotypic screen to identify compounds as novel biological tools and starting points for drug discovery projects and we disclose the first small molecules to proliferate human primary EPDCs.

INTRODUCTION The adult mammalian heart has limited endogenous ability to self-repair after injury and with the advances in treatment options for post-myocardial infarction (MI) along with rising life expectancy, an increasing number of patients survive to progress to heart failure. Once heart failure develops, treatment options are limited to therapies that can merely slow or stop the spread of damage without the ability to restore normal cardiac function. The high rates of morbidity and mortality result in a huge burden on healthcare systems in developed countries1 and the dismal prognosis for patients with heart failure has prompted efforts to develop regenerative treatment strategies.2 In recent years, epicardium-derived cells (EPDCs) have been shown to be an important cell type, specifically in signaling for heart development and post-MI heart


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3 of 18 repair.3 During heart development, EPDCs also contribute to formation of different cardiac cell types and secrete important trophic factors for myocardial maturation.3, 4 In the adult heart, EPDCs are quiescent under normal conditions and become activated following myocardial injury. In zebrafish, organ-wide activation of the epicardium has been shown to be critical for heart regeneration in response to injury.5 Following MI in mice, EPDCs proliferate, undergo an epithelial-to-mesenchymal transition, primarily characterized by expression of Wilm’s Tumor protein 1 (WT-1), migrate into the myocardium, and differentiate into predominately smooth muscle cells and fibroblasts as well as a smaller percentage of endothelial cells.6 In addition, when primed with thymosin beta-4, EPDCs have been shown to proliferate and improve cardiac function post-MI,7 which supports the general approach to investigate EPDC proliferation. In addition to contributing to the formation of new cardiac cells it has been demonstrated that EPDCs secrete important paracrine growth factors, such as VEGF-A, FGF2 and PDGF-CC, which have been shown to promote growth of blood vessels, reduce infarct size, and improve heart function in a mouse model of acute MI.4 Despite growing interest in EPDCs for cardiac regeneration, the molecular mechanisms regulating EPDC activation, proliferation and/or differentiation are poorly understood, warranting further investigation. In areas of novel and complex pharmacology such as stem cell and regenerative biology, where there is incomplete knowledge of potential pathways and targets for disease intervention, screening compounds in an unbiased fashion using high content phenotypic screening with human disease relevant cells is particularly attractive. In recent years phenotypic screening has seen a renaissance with reports demonstrating the importance of phenotypic screening to discover novel, first-in-class drugs.8, 9 Screening in a target-independent manner provides the opportunity to identify compounds that (i) modulate novel targets; (ii) modulate known targets which are unknown in this disease context; (iii) hit multiple targets or biological pathways.10, 11 Hence, this approach enables the discovery of novel biological targets and new compounds, as well as compounds showing new mechanisms of action or polypharmacology. The cells most representative of human physiology are human primary cells and should be used where possible in



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4 of 18 phenotypic screening campaigns.12 However, a major challenge in this context is the ability to isolate sufficient quantities of primary cells from patients, which prohibits screening of large compound collections. With this limitation, compounds must be carefully selected to include modulators of as many biological targets as possible, and to broadly sample chemical space. To identify compounds that enhance proliferation of human EPDCs, we developed a robust phenotypic screening assay in primary EPDCs isolated from human adult heart during cardiac surgery6 and screened a selected library of compounds. This phenotypic screening approach identified chemically diverse compounds that increased proliferation of EPDCs as measured by nuclear count. Careful design of the screening cascade allowed clusters of compounds to be identified which induced proliferation of EPDCs from different donors, did not alter their ability to differentiate into other essential cell types, and importantly did not cause proliferation of human primary cardiac fibroblasts (CFs). This is the first report of small molecules which enhance proliferation of human EPDCs. As such these compounds are valuable tools for understanding the biology of EPDCs, their link to human cardiac regeneration and also starting points to discover novel regenerative therapies for heart failure patients.

RESULTS AND DISCUSSION Cell source Primary human epicardial cells were isolated from four patients and expanded for three passages during which they underwent epithelial-to-mesenchymal transition and became EPDCs.3, 6 EPDCs are characterized by their spindle-shaped morphology and expression of various markers, including WT-1, with an absence of expression of differentiation markers such as α-SMA. This phenotype was verified in all donors prior to further expansion and banking.


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5 of 18 EPDCs can maintain their phenotype over multiple passages In order to set up a screen and test a sizeable number of compounds, it was necessary to expand the initial batch of EPDCs. Primary cells have a limited expansion capacity, so a key aspect was quality control to ensure that the cell banks generated for screening were robust and maintained the EPDC phenotype of interest. Cell yield, growth rate, and cell/nuclear morphology were monitored throughout the expansion process (Figure 1a, b, d). In addition, cells taken at different passages were examined for WT-1, a key marker of EPDCs (Figure 1b–d). The cells expanded robustly up to passage nine, but at later passages (particularly at passage twelve and beyond) the growth rate began to slow and the morphology of the cells changed, with the cells and nuclei both becoming larger while the expression of WT-1 decreased (Figure 1d). Based on this data, it was determined that EPDCs should be used for screening at passage nine or earlier to ensure that the hits identified would be active on cells that maintained the required phenotype.

Development of a proliferation assay Securing a robust and reproducible assay set up is as critical as using the right cells for a screen and is key to generating relevant data. A phenotypic screen can give many possible readouts but as the primary goal was to identify molecules with an ability to proliferate the cells, nuclear count was selected as the main parameter as a measure of cell number. Development of a phenotypic screen using primary cells is particularly challenging especially in the absence of known positive controls, even if the endpoint is fairly straightforward. During assay development several parameters were optimized, including time for endpoint readout, plate type, seeding density, method of cell plating, and compound dosing and fixation protocols. Initially, a growth media (EGM2) with a cocktail of growth factors (VEGF, IGF, FGF and EGF) was used as a starting condition to show that EPDCs can proliferate robustly in a 384 well plate format. When optimal conditions for each of the above parameters were determined, a set of compounds was screened to validate the robustness and reproducibility of the assay as well as to attempt to find a



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6 of 18 small molecule with a high pro-proliferative effect to replace the growth media cocktail as a positive control. At the conclusion of assay development we secured an assay with a robust signal to background of 1.4, Z'13 of 0.4 and good reproducibility (Figure 2a–c).

Screening of compounds The technical and biological challenges with isolating EPDCs from human patient biopsies and subsequently expanding to considerable cell numbers made a diversity screen with large numbers of compounds unfeasible. Instead, the EPDC proliferation assay was used to screen a small molecule compound library of 7400 compounds. It consisted of two specifically selected sets of compounds. The main set (6900 compounds) was designed to consist of structurally diverse molecules which are known to act on certain biological targets (i.e. have an annotated mechanism of action) to maximize the coverage of known biological target space. This compound set consists of marketed drugs, internal AstraZeneca compounds optimized for specific targets including clinical candidates and lead molecules from preclinical drug discovery projects and drug project compounds which have shown potent activity at a target within broad secondary pharmacology14 or kinase profiling panel screens. In addition, this compound set also contained compounds identified from literature, including clinical candidates and chemical probes for novel targets sourced either from external vendors or synthesized in-house, to further enhance the biological target coverage and activity of the compound library. In total the 6900 member library contained representative molecules with activity values (Ki, EC50 or IC50) of less than 100 nM against more than 1500 biological targets across different target classes including GPCRs, nuclear hormone receptors, ion channels and enzymes, of which kinases constituted a major part (Figure 2d). The second component of the screening set was a selection of 500 compounds drawn from literature with a reported ability to modulate the phenotype of stem or primary cells. This includes compounds inducing proliferation or differentiation,15, 16, 17, 18, 19 compounds affecting key developmental pathways (for example


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7 of 18 Wnt, TGFβ, Notch, or Hedgehog signaling pathways), or compounds acting on epigenetic targets such as histone deacetylase or bromodomain inhibitors. In contrast to the 6900 member library, this 'stem cell set' also included molecules with unknown mechanisms of action. The 7400 molecules were screened in three point concentration response (CR) at 0.1, 1.0 and 10 µM in duplicate measuring proliferation of human EPDCs obtained from a single donor. From the primary screen 220 compounds were identified that had a measured nuclear count above the activity threshold at 3×SD of the neutral control in at least one of the three concentrations screened (Figure 3a).

Compounds inducing proliferation of human primary EPDCs Hit molecules identified in the primary screen were followed up in ten point CR which confirmed 50% as active hits (110 compounds). One challenge in performing screens with limited numbers of compounds is that many of the primary hits are often chemical singletons. This contrasts to regular target-based highthroughput screening campaigns where typically greater than one million compounds are screened and clusters of chemically similar compounds are often identified which show a range of activity and early structure–activity relationships (SAR) leading to greater confidence in the strength of the chemical hit as a starting point. In our EPDC screen, chemical clustering of the 110 confirmed hits revealed fifteen singletons and ten chemical clusters consisting of between two and 20 compounds. To follow up the singleton hits or very small compound clusters, chemically similar compounds20 from the AstraZeneca compound collection were identified. These were selected based on multiple fingerprint methods using extended connectivity fingerprints, an in-house implementation of the Daylight fingerprints and a textbased molecular similarity searching.21, 22 In parallel, we investigated the annotated biological activity of the hit molecules to generate early hypotheses for biological targets which may be driving the proliferative phenotype.23 Subsequently diverse compounds were selected which modulate the enriched biological targets. These chemical and biological target approaches provided an additional 90 compounds which



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8 of 18 were screened in the EPDC proliferation assay, giving a total of 200 compounds being progressed in the screening cascade (Figure 3a). Evaluation of the hit molecules included the identification of two structurally diverse compounds, 1 and 2, that showed different levels of potency and efficacy in the EPDC proliferation assay. Activity is reported using the negative logarithm of the lowest effective concentration (pLEC),24 pEC50, and maximum effect (%). Compounds 1 and 2 display a pLEC of 7.0 and 5.8 with a maximum proliferative effect of 93% and 39% respectively (Tables 1 and 2). After the initial primary screen, no close structural analogs of compounds 1 and 2 were identified in the AstraZeneca compound collection. Hence, analogs to these two compounds were synthesized (for synthetic procedures see supporting information). The synthesis of 1 and some derivatives has been described previously by Shen et al.25 Briefly, the central core of compound 1 could be modified from a methylene amide to a urea moiety as shown in 3 (Figure 4). To rapidly build the chemical cluster, variations were made by replacing the methylpiperazine group in compound 1 or 3 with alternative amine functionality using reductive amination chemistry to afford derivatives 4–8 (Figure 4, Table 1, SI Scheme S1–S2). These derivatives were tested in the EPDC proliferation assay and, similarly to 1, compounds with larger diamine substituents (4–7) were shown to have very high potency and efficacy. In contrast, the smaller dimethylamine 8 was inactive. Compounds 3 and 6 were ten-fold more potent compared to compound 1 with similar lipophilicity (logD). To develop SAR around compound 2 we varied the amide substituent as well as the 1N- and 2-positions of the benzimidazole ring (Figure 4). The amide substituent was varied in the last step of the reaction sequence through coupling the benzimidazole acid intermediate with a range of substituted anilines to give compounds 9–14 (Figure 4, Table 2, SI Scheme S3). The substituents on the benzimidazole ring, in both the 1N- and 2-positions could be investigated by utilizing 1,1,1-trimethoxyethane or


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9 of 18 triethylorthopropionate in the formation of the benzimidazole ring to synthesise 2 and 15 respectively or by nucleophilic aromatic substitution (for example, 16) (Table 2, SI Scheme S4 and S5). Starting with the phenylamide, small substituents such as halogen or methyl in the meta- or para-position of the phenyl ring provided more potent compounds than 2 with pLEC values ranging from 6.8–7.0 (9, 11 and 12). Phenyl derivatives (10 and 13) were equipotent with 2. A methyl- or benzyl-amide were inactive (data not shown). Replacing the phenyl ring with various heterocycles provided less lipophilic compounds but the maximum effect was below the activity threshold of 30% (e.g. pyrazine amide 14). Ethyl substitution in the 2-position of the benzimidazole, in place of the methyl group in 2, rendered an inactive molecule (15), whereas an 1N-n-propyl substituent as in 16 provided a very potent and efficacious compound. A 1N-phenyl substituent in combination with the 2,4-difluorophenyl amide (17) also gave good activity. These results confirm this chemical cluster as having excellent potency on driving proliferation of human EPDCs, albeit with lower efficacy than compound 1.

Cardiac fibroblast counterscreen A major response of the heart after MI is to increase fibrosis via matrix deposition from activated myofibroblasts so we therefore designed a counterscreen to eliminate hit molecules with undesirable effects on proliferation of cardiac fibroblasts (CFs). To address this, the confirmed actives on the EPDCs were progressed to the human CF proliferation counterscreen where cell number as measured by nuclear count was the primary endpoint. This phenotypic screening strategy identified compounds with different activity profiles on EPDCs and CFs, highlighting the importance of designing an effective screening cascade to remove the more pan-proliferative molecules. For example, compound 1 gave a profile that proliferated EPDCs both with high efficacy (93%) and potency (pLEC=7.0) but also proliferated CFs (pLEC=6.3, max effect=59%) (Table 1, Figure 3b), and this pan-proliferative profile was true of many active compounds in this chemical cluster including 1, 3 and 4. In contrast, compound 9 (an analog of 2)



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10 of 18 induced clear proliferation of EPDCs (pEC50=6.0, pLEC=5.8), albeit with lower efficacy (max effect=39%), but did not increase nuclear count of the CFs above the activity threshold (Table 2, Figure 3c). As this chemical cluster did not increase cell count of CFs, it appears more specific towards EPDCs. In total the CF counterscreen eliminated 20 compounds from seven different clusters or singletons. Due to its differential proliferation profile with activity on EPDCs and not CFs, as well as its interesting annotated biology and clean secondary pharmacology profile (see later), compound 2 was selected and taken forward. In addition, due to its higher efficacy and effects on both EPDCs and CFs, 1 was also taken forward as an important tool compound to investigate the biology of both cell types.

Cross-donor validation and differentiation of EPDCs after expansion Primary cells, although more biologically relevant, have a greater degree of variability than cell lines which raises concerns about donor-to-donor variation. To address this, additional testing was performed across several EPDC donors to increase confidence in the translatability of the selected compounds 1 and 2. This confirmed that the hits that we progressed were active across four different donors and increased EPDC proliferation (Figure 5a). Some variability was seen in the efficacy of 1 at the concentration tested, which is to be expected with primary cells from different donors. As EPDCs are a population of progenitor cells, a key function is their ability to differentiate into cardiac cell populations, with the default lineages being smooth muscle cells and fibroblasts. The primary goal of our screen was to identify compounds that proliferate EPDCs without negatively influencing differentiation, and it was therefore important to ensure the cells maintained the progenitor phenotype after compound treatment. Hence, compounds were analyzed for their effects on WT-1 expression in EPDCs after expansion and it was confirmed that they did not alter WT-1 expression levels (data not shown). A critical secondary assay was to investigate the differentiation capacity of the cells after compound-induced expansion. Hence, the leading compounds which progressed through the CF


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11 of 18 counterscreen were first used to expand the cells and after expansion the cells were stimulated with BMP2 to induce differentiation to smooth muscle cells and fibroblasts.26, 27 Compounds 1 and 2 showed similar levels of differentiation as the control cells after addition of BMP2, as demonstrated by α-SMA staining (Figure 5b–e). This provided additional support that the compounds were not changing the progenitor phenotype of the EPDCs or inducing differentiation down other cell lineages. Investigation of targets driving the phenotypic effect After confirming activity of the hit molecules and demonstrating that these could be grouped into chemical clusters with an ability to proliferate EPDCs, we turned our attention to their biological annotation. As highlighted, most compounds selected for screening were associated with known biological activity against a certain target or targets. While this does not exclude the possibility that the compound may also be active against additional targets, it provides a starting point to investigate a potential mechanism of action. Compounds 1 and 3 have previously been reported to induce proliferation of rodent and human β-cells.25 In that disclosure, a chemoproteomics pull-down experiment followed by a shRNA knockdown approach suggested that these compounds drive β-cell proliferation through inhibition of nuclear factor kappa-B kinase subunit epsilon (IKBE), and ErbB3-binding protein 1 (PA2G4). Those experiments demonstrated that in R7T1 cells, PA2G4 knockdown using shRNA resulted in a 1.8-fold increase in cell viability, although IKBE knockdown with shRNA was not tested.25 To examine whether inhibiting these targets induced proliferation of human EPDCs, we performed knockdown experiments using siRNA against IKBE and PA2G4. Neither knockdown of IKBE or PA2G4 resulted in an increase in EPDC cell proliferation (Figure 6a). This result may be due to differences in measurement technology between the two studies (an increase in cell viability may not necessarily translate into an increase in cell proliferation), or due to inherent differences between EPDCs and R7T1 cells. Hence, the targets implicated to drive proliferation of β-cells with 1 could not be confirmed as the targets driving proliferation of human EPDCs, highlighting the complexity of the biology under investigation.



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12 of 18 To further assess the selectivity and find alternative putative targets driving proliferation, 1 was screened in biochemical assays against 353 kinases (Thermo Fisher Scientific) and shown to inhibit many kinases (71 with > 75% inhibition at 1 µM compound concentration, SI Figure S1). To verify the single point activity data, 15 kinases with an inhibition greater than 75% were selected and full IC50 curves were determined. Inhibition was confirmed with IC50 values less than 1 µM for all 15 kinases (SI Table S1). Furthermore, compound 1 was profiled in a secondary pharmacology panel14 consisting of 147 targets from a range of target classes. The compound was shown to inhibit additional kinases with IC50 values of < 1 µM and demonstrated binding affinity or functional activity (Ki, IC50 or EC50 values) of < 10 µM towards 37 additional targets. This broad activity profile highlighted that this compound was promiscuous against a range of targets and had the potential to modify multiple biological pathways (SI Table S1). So although we were unable to demonstrate that the targets driving EPDC proliferation were the same as the targets driving β-cell proliferation, the selectivity profile of 1 suggested that the compound could have elicited its effect on EPDCs by a completely different mechanism or by polypharmacology. The chemical cluster represented by 2 had a very different profile. Compounds 2 and 9 have previously been shown to promote expansion of CD34+ hematopoietic stem cells by antagonism of the aryl hydrocarbon receptor (AhR).28, 29 To investigate further, novel synthesized members of this compound cluster were included in an AhR antagonism assay, indicating activity against AhR via reduced expression of the downstream marker cytochrome P450 CYP1A1/2 protein in dioxin treated HepG2 cells (Table 2).30, 31

The compounds showed no agonistic properties in cells without dioxin treatment but did indeed display

activity as antagonists (e.g. 2 displays a pIC50=6.0). To build confidence that AhR is a key target driving proliferation of EPDCs, structurally diverse molecules which have been reported to display antagonistic activity against AhR, were tested in the EPDC assay. These molecules included StemRegenin 1 (18)28, 29 and 1928 (SI Table S2), which both induced proliferation of EPDCs (pEC50=6.2, pLEC=7.1, max effect=50% for 18 and pLEC=6.4, max effect=38% for 19) and 18 also showed potent activity in the AhR antagonist assay. With a reasonable target hypothesis in place we performed a knockdown experiment


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13 of 18 using siRNA against AhR. Interestingly, this did not induce proliferation of the cells (Figure 6a). Complete knockdown at the protein level was confirmed (Figure 6b, Figure S2 with GAPDH loading control), which suggests that whilst these molecules have activity against the downstream target CYP1A1/2 in HepG2 cells, a reduction of AhR protein level alone is not sufficient to drive the proliferative phenotype in EPDCs, which could mean that the compounds inhibit additional targets rather than directly modulate AhR. To check for general selectivity and identify possible alternative targets, compound 2 was profiled against 269 kinases (Merck-Millipore) but showed < 50% inhibition at 10 µM compound concentration across the whole panel (Figure S3). Compound 2 was also profiled in a secondary pharmacology panel14 consisting of 147 targets and only bound to three targets with an affinity of less than 10 µM with the most potent being 5-HT2b with a Ki value of 2.0 µM (SI Table S3). Hence, in contrast to 1, compound 2 shows very little activity in these target panels. Taken together, the lack of potent alternative targets identified in the kinase and pharmacological panels as well as the structural diversity of the AhR antagonists driving proliferation of EPDCs (e.g. 2, 18, and 19), suggest that the proliferation phenotype may not be completely independent of AhR antagonism. This could be explained by polypharmacology or an effect on AhR unrelated to reduction in protein levels alone. Further work is ongoing to understand the detailed mechanism of action of these molecules.

Conclusion This is the first report of small molecules which enhance the proliferation of human primary EPDCs isolated from patients. As such, these compounds are valuable tools for understanding the biology of EPDCs and their link to human cardiac regeneration, and also represent starting points to discover new regenerative therapies for heart failure patients. The origin of the two compound cluster representatives 1 and 2 was a dedicated small 'stem cell set', clearly demonstrating the usefulness of text mining and chemoinformatic approaches to compile informed



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14 of 18 compound sets for phenotypic screening. Based on the effects on CFs and the promiscuity against many different targets, 1 and the associated chemical cluster will not be progressed from a drug discovery perspective. Rather, these compounds will be utilized as valuable tools to develop future screening cascades and to build further knowledge around EPDC biology. The chemical cluster around 2 is more appealing as it does not induce proliferation of CFs and is very clean in target panel screens. Work is ongoing to improve the compound properties of this series, in particular drug metabolism and pharmacokinetic properties to enable effective in vivo experiments, as well as to understand more about its precise mechanism of action driving EPDC proliferation, including the potential role of AhR antagonism. The importance of investigating the annotated targets of compounds is highlighted as the biology under investigation is complex and different cell populations can have varying responses to the same stimulus, as was found with the different results between PA2G4 knockdown effects in EPDCs and R7T125 cells. In addition, this work demonstrates that compounds can be identified that have differential proliferation profiles, shown here on EPDCs and CFs, highlighting the possibility to identify and exploit cell-type specific pathways and phenotypes.

METHODS For all methods refer to Supporting Information for more details.

ABBREVIATIONS AhR, aryl hydrocarbon receptor; CF, cardiac fibroblasts; CR, concentration response; DAPI, 4',6diamidino-2-phenylindole; EPDC, epicardium-derived cells; IKBE, nuclear factor kappa-B kinase subunit epsilon; iPSC, induced pluripotent stem cell; pLEC, negative logarithm of the lowest effective


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15 of 18 concentration; MI, myocardial infarction; PA2G4, ErbB3-binding protein 1; SAR, structure–activity relationship; WT-1, Wilm’s Tumor protein 1.

ASSOCIATED CONTENT Supporting information Supporting Information Available: This material is available free of chare via the internet, including complete list of synthetic procedures and compound characterization for the synthesis of compounds 1– 17, materials, experimental procedures, supplementary tables S1–S3, and figures S1–S3.

AUTHOR INFORMATION Notes: The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors would like to acknowledge M. Polla and E. Wellner for assistance with screening set selection and hit identification data analysis, L. Roth for expansion of the cells, L. von Sydow for performing HRMS and K. Nilsson for help with NMR measurements, and U. Karlsson and S. Peel for help with development and implementation of the EPDC proliferation assay.



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16 of 18 REFERENCES

(1) Roger, V. L. (2013) Epidemiology of heart failure. Circ. Res. 113, 646-659. (2) Lin, Z., and Pu, W. T. (2014) Strategies for cardiac regeneration and repair. Science Translational Medicine. 6, 239rv1-239rv1. (3) Gittenberger-de Groot, A. C., Winter, E. M., and Poelmann, R. E. (2010) Epicardium-derived cells (EPDCs) in development, cardiac disease and repair of ischemia. J. Cell. Mol. Med. 14, 1056-1060. (4) Zhou, B., Honor, L. B., He, H., Ma, Q., Oh, J., Butterfield, C., Lin, R., Melero-Martin, J., Dolmatova, E., Duffy, H. S., Gise, A. v., Zhou, P., Hu, Y. W., Wang, G., Zhang, B., Wang, L., Hall, J. L., Moses, M. A., McGowan, F. X., and Pu, W. T. (2011) Adult mouse epicardium modulates myocardial injury by secreting paracrine factors. J. Clin. Invest. 121, 1894-1904. (5) Lepilina, A., Coon, A. N., Kikuchi, K., Holdway, J. E., Roberts, R. W., Burns, C. G., and Poss, K. D. (2006) A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell (Cambridge, MA, U. S. ). 127, 607-619. (6) Bax, N. A. M., Van Oorschot, A. A. M., Maas, S., Braun, J., Van Tuyn, J., De Vries, A. A. F., Gittenbergerde Groot, A. C., and Goumans, M. J. (2011) In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGFβ-signaling and WT1. Basic Res. Cardiol. 106, 829-847. (7) Smart, N., Dubé, K. N., and Riley, P. R. (2013) Epicardial progenitor cells in cardiac regeneration and neovascularisation. Vascular Pharmacology; Recent Advances in Cardiovascular Biology and Translational Medicine. 58, 164-173. (8) Eder, J., Sedrani, R., and Wiesmann, C. (2014) The discovery of first-in-class drugs: Origins and evolution. Nat. Rev. Drug Discovery. 13, 577-587. (9) Swinney, D. C., and Anthony, J. (2011) How were new medicines discovered? Nat. Rev. Drug Discovery. 10, 507-519. (10) Vincent, F., Loria, P., Pregel, M., Stanton, R., Kitching, L., Nocka, K., Doyonnas, R., Steppan, C., Gilbert, A., Schroeter, T., and Peakman, M. (2015) Developing predictive assays: The phenotypic screening "rule of 3". Sci. Transl. Med. 7, 1-5. (11) Eggert, U. S. (2013) The why and how of phenotypic small-molecule screens. Nat. Chem. Biol. 9, 206209. (12) Engle, S. J., and Vincent, F. (2014) Small molecule screening in human induced pluripotent stem cellderived terminal cell types. J. Biol. Chem. 289, 4562-4570. (13) Zhang, J., Chung, T. D. Y., and Oldenburg, K. R. (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Journal of Biomolecular Screening. 4, 6773.


Page 17 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


17 of 18 (14) Bowes, J., Brown, A. J., Hamon, J., Jarolimek, W., Sridhar, A., Waldron, G., and Whitebread, S. (2012) Reducing safety-related drug attrition: The use of in vitro pharmacological profiling. Nat Rev Drug Discov. 11, 909-922. (15) Schade, D., and Plowright, A. T. (2015) Medicinal chemistry approaches to heart regeneration. J. Med. Chem. 58, 9451-9479. (16) Plowright, A. T., Engkvist, O., Gill, A., Knerr, L., and Wang, Q. (2014) Heart regeneration: Opportunities and challenges for drug discovery with novel chemical and therapeutic methods or agents. Angew. Chem. , Int. Ed. 53, 4056-4075. (17) Davies, S. G., Kennewell, P. D., Russell, A. J., Seden, P. T., Westwood, R., and Wynne, G. M. (2015) Stemistry: The control of stem cells in situ using chemistry. J. Med. Chem. 58, 2863-2894. (18) Drowley, L., Koonce, C., Peel, S., Jonebring, A., Plowright, A. T., Kattman, S. J., Andersson, H., Anson, B., Swanson, B. J., Wang, Q. D., and Brolen, G. (2016) Human induced pluripotent stem cell-derived cardiac progenitor cells in phenotypic screening: A transforming growth factor-β type 1 receptor kinase inhibitor induces efficient cardiac differentiation. Stem Cells Transl Med. 5, 164-174. (19) Billin, A. N., Bantscheff, M., Drewes, G., Ghidelli-Disse, S., Holt, J. A., Kramer, H. F., McDougal, A. J., Smalley, T. L., Wells, C. I., Zuercher, W. J., and Henke, B. R. (2016) Discovery of novel small molecules that activate satellite cell proliferation and enhance repair of damaged muscle. ACS Chem. Biol. 11, 518-529. (20) Shanmugasundaram, V., Maggiora, G. M., and Lajiness, M. S. (2005) Hit-directed nearest-neighbor searching. J. Med. Chem. 48, 240-248. (21) Grant, J. A., Haigh, J. A., Pickup, B. T., Nicholls, A., and Sayle, R. A. (2006) LINGOs, finite state machines, and fast similarity searching. J. Chem. Inf. Model. 46, 1912-1918. (22) Kogej, T., Engkvist, O., Blomberg, N., and Muresan, S. (2006) Multifingerprint based similarity searches for targeted class compound selection. J. Chem. Inf. Model. 46, 1201-1213. (23) Bornot, A., Blackett, C., Engkvist, O., Murray, C., and Bendtsen, C. (2014) The role of historical bioactivity data in the deconvolution of phenotypic screens. J. Biomol. Screening. 19, 696-706. (24) Bosveld, A. T. C., de Bie, Paul A. F., van, d. B., Jongepier, H., and Klomp, A. V. (2002) In vitro EROD induction equivalency factors for the 10 PAHs generally monitored in risk assessment studies in the netherlands. Chemosphere. 49, 75-83. (25) Shen, W., Tremblay, M. S., Deshmukh, V. A., Wang, W., Filippi, C. M., Harb, G., Zhang, Y., Kamireddy, A., Baaten, J. E., Jin, Q., Wu, T., Swoboda, J. G., Peters, E. C., Cho, C. Y., Li, J., Laffitte, B. A., McNamara, P., Glynne, R., Wu, X., Herman, A. E., and Schultz, P. G. (2013) Small-molecule inducer of β cell proliferation identified by high-throughput screening. J. Am. Chem. Soc. 135, 1669-1672. (26) Grieskamp, T., Rudat, C., Lüdtke, T. H. -., Norden, J., and Kispert, A. (2011) Notch signaling regulates smooth muscle differentiation of epicardium-derived cells. Circ. Res. 108, 813-823.



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18 of 18 (27) van Tuyn, J., Atsma, D. E., Winter, E. M., van, d. V., Pijnappels, D. A., Bax, N. A. M., Knaaen-Shanzer, S., Gittenberger-de Groot, A. C., Poelmann, R. E., van, d. L., van, d. W., Schalij, M. J., and de Vries, Antoine A. F. (2007) Epicardial cells of human adults can undergo an epithelial-to-mesenchymal transition and obtain characteristics of smooth muscle cells in vitro. Stem Cells (Durham, NC, U. S. ). 25, 271-278. (28) Bouchez, L. C., Boitano, A. E., de Lichtervelde, L., Romeo, R., Cooke, M. P., and Schultz, P. G. (2011) Small-molecule regulators of human stem cell self-renewal. ChemBioChem. 12, 854-857. (29) Boitano, A. E., Wang, J., Romeo, R., Bouchez, L. C., Parker, A. E., Sutton, S. E., Walker, J. R., Flaveny, C. A., Perdew, G. H., Denison, M. S., Schultz, P. G., and Cooke, M. P. (2010) Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science (Washington, DC, U. S. ). 329, 1345-1348. (30) Whitlock, J. P., Jr. (1989) The control of cytochrome P-450 gene expression by dioxin. Trends Pharmacol. Sci. 10, 285-288. (31) Garside, H., Stewart, A., Brown, N., Cooke, E., Graham, M., and Sullivan, M. (2008) Quantitative analysis of aryl hydrocarbon receptor activation using fluorescence-based cell imaging. A highthroughput mechanism-based assay for drug discovery. Xenobiotica. 38, 1-20.


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142x80mm (300 x 300 DPI)



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Figure 1: EPDC scale up and characterization. (a) Brightfield images of early (P5), mid (P9), and late (P12) passages of the EPDCs. (b) Nuclei staining with DAPI (upper) and WT-1 (lower). (c) Control staining of iPSC with DAPI and WT-1. (d) Percent WT-1 positive cells at harvest for each passage with replicates. 131x112mm (300 x 300 DPI)


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Figure 2: Setup of phenotypic screen. (a) Outline of the screening program developed to identify small molecules that induce EPDC proliferation. Main readout is nuclear count. (b) Representative distribution of positive (maximal) and neutral controls in the EPDC proliferation assay with signal to background of 1.4 and Z’ = 0.4. (c) Correlation between biological replicated indicated that the assay is robust across screening occasions. Data points represent means of normalized cell count and line represents the correlation of means (n=2) of biological replicates during validation of the assay. (d) Coverage of target classes in the annotated set. 129x76mm (300 x 300 DPI)



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Screening cascade and hit progression. (a) Illustration of screening cascade and outcome of each step. Concentration response effects for compounds 1 (b) and 9 (c) giving different proliferation profiles. Proliferation measured as nuclei count, normalized to % effect from the maximal and neutral controls for both EPDCs and CFs, where compound 1 shows unspecific proliferation and compound 9 only proliferates EPDCs (the CFs are within the variation of the assay). Data points represent the mean of biological replicates, where each biological replicate consists of technical duplicates, with error bars = SEM. (b: EPDC n=14, CF n=2. C: EPDC n=3, CF n=2). 137x149mm (300 x 300 DPI)


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Table 1. Activity on EPDC and CF proliferation of cyclopropanecarboxamide derivatives

Compound

X

R1

EPDC pEC50a /

EPDC Max effecta,c

CF pLECa,b / Max effectc

logD7.4

93%±15%

6.3±0.4

4.0

pLECa,b 125

CH2

Active-NCd / 7.0±0.2

59%±6%

325

NH

8.0±0.1 /

62%±22%

8.0±0.2 4

CH2

Active-NCd/

NH

Active-NCd/

83%±11%

NH

Active-NCd /

45%±21%

NH

Active-NCd / 7.0±0.03

3.6

NAe/

4.0

4%±2%

79%±30%

7.9±0.1

7

5.9±0.2 50%±5%

7.2±0.3

6

3.9

32%±7%

6.5±0.3

5

7.3±0.04

NAe/

4.1

13%±4%

66%±12%

NAe/

5.1

12%±10% 8

CH2

NAe /

-2%

NAe


NAe/ 13%±8%

4.0


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a

The proliferation pEC50, pLEC and top effect reported are averages from n2 biological replicates

where each replicate includes technical duplicates. b

LEC is determined as the concentration where the compound reaches above the activity cut off set as

3×SD=30% for the EPDC assay. c

Maximum effect reached relative to the positive control.

d

Active NC=Active no curve fit, the compound does not reach a plateau but displays proliferation

above the activity cut off set as 3×SD=30%. e

NA=Not active


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Table 2. Activity on EPDC proliferation and AhR antagonism of benzimidazole derivatives

Compound

R1

R2

R3

EPDC pEC50a /

EPDC Max effecta,c

AhR antagonism pIC50

logD7.4

39%±7%

6.1±0.7

2.5

68%±17%

4.0

6%

NAe

1.5

19%

NAe

2.9

pLECa,b 228

Me

Et

6.9±0.1 / 5.8±0.9

928

Me

Et

6.8±0.2 / 7.0±0.3

10

Me

Et

5.8±0.2 / 5.8±0.3

11

Me

Et

6.9±0.2 / 6.8±0.1

12

Me

Et

ActiveNCd / 6.9±0.1

13

Me

Et

6.3±0.2/ 6.2±0.2

14

Me

Et

NAe / NAe

15

Et

Et

NAe / NAe



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16

Me

n-Pr

6.7±0.2 / 6.8±0.4

64%±10%

6.0±0.4

3.1

17

Me

Ph

ActiveNCd/

59%±17%

6.1±0.7

-

5.9±0.1

a

The EPDC proliferation pEC50, pLEC and top effect reported are averages from n2 biological

replicates where each replicate include technical duplicates. b

LEC is determined as the concentration where the compound reaches above the activity cut off set as

3×SD=30% for the EPDC assay. c

Maximum effect reached relative to the positive control.

d

Active NC=Active no curve fit, the compound does not reach a plateau but displays proliferation

above the activity cut off set as 3×SD=30%. e

NA=Not active


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Figure 4: Structures of hit molecules 1 and 2 and emerging structure-activity relationships. 190x107mm (300 x 300 DPI)



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Figure 5: Followup of hits on EPDC phenotype. (a) EPDCs from four different donors were treated with 1 µM compound 1 or 3 µM compound 2 to examine effects on proliferation. Differentiation down smooth muscle/fibroblast lineages after compound-induced expansion. Basal levesl of α-SMA (green) expression before (b) and after (c) BMP2 treatment. EPDCs proliferation with compound 2 (d) and compound 1 (e) and then treated with BMP2 show no change in differentiation capacity. 253x451mm (600 x 600 DPI)


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Figure 6: Exploration of putative target mechanism of action. (a) Knocking down AhR, IKBKE, or PA2G4 in EPDCs did not induce cell proliferation, suggesting involvement of additional components in the mechanism of action of the proliferation-inducing compounds annotated as inhibiting these targets. (b) Western blot confirmation of AhR presence and knockdown in EPDCs at 72h and 96h post siRNA transfection. 100x47mm (300 x 300 DPI)


Phenotypic Screen for Cardiac Regeneration ... - ACS Publications

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