Chemogenomic Profiling of Endogenous PARK2 Expression Using

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Chemogenomic profiling of endogenous PARK2 expression using a genome-edited coincidence reporter Samuel A. Hasson, Adam I. Fogel, Chunxin Wang, Ryan MacArthur, Rajarshi Guha, Sabrina Heman-Ackah, Scott Martin, Richard J. Youle, and James Inglese ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb5010417 • Publication Date (Web): 17 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Chemogenomic profiling of endogenous PARK2 expression using a genome-edited coincidence reporter

Samuel A. Hassona,e,§, Adam I. Fogela,e, Chunxin Wanga, Ryan MacArthurb, Rajarshi Guhab, Sabrina Heman-Ackahc,†, Scott Martinb, Richard J. Youlea, and James Ingleseb,d,* a

b

National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892, National Center for c

Advancing Translational Sciences, Rockville, MD 20850, NIH Center for Regenerative Medicine, National d

Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland 20892, National e

Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, These authors contributed equally. §

current address: Pfizer Inc., Cambridge, MA, 02139

†

current address: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3QX, UK *To whom correspondence should be addressed. E-mail: [email protected], Phone: 301217-5723, Fax: 301-217-5736

Keywords: Drug discovery, Parkinson’s disease, HTS, qHTS, genome editing, reporter gene, coincidence reporter, luciferase, chemical library, parkin, mitochondrial quality control Abbreviations: qHTS, quantitative high throughput screening; FLuc, firefly luciferase; NHR, nuclear hormone receptor; NLuc, NanoLuciferase; HDR, Homology-directed repair ; ∆OTC, truncated Ornithine transcarbamylase Running Title: Coincidence Reporter Screen for PARK2 Expression

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Abstract

Parkin, an E3 ubiquitin ligase, is a central mediator of mitochondrial quality control and is linked to familial forms of Parkinson’s disease (PD). Removal of dysfunctional mitochondria from the cell by Parkin is thought to be neuroprotective, and pharmacologically increasing Parkin levels may be a novel therapeutic approach. We used genome editing to integrate a coincidence reporter into the PARK2 gene locus of a neuroblastoma-derived cell line, and developed a quantitative high-throughput screening (qHTS) assay capable of accurately detecting subtle compound-mediated increases in endogenous PARK2 expression. Interrogation of a chemogenomic library revealed diverse chemical classes that up-regulate PARK2 transcript, including epigenetic agents, drugs controlling cholesterol biosynthesis, and JNK inhibitors. Use of the coincidence reporter eliminated wasted time pursuing reporter-biased false positives accounting for ~⅔ of the actives, and coupled with titration-based screening greatly improves the efficiency of compound selection. This approach represents a strategy to revitalize reporter-gene assays for drug discovery.

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Introduction Mitochondria are dynamic organelles at the center of metabolism and the biosynthesis of lipids and heme. Mitochondrial dysfunction is associated with aging and numerous diseases, including neurodegenerative disorders such as Parkinson’s disease (PD) 1. When mitochondria lose membrane potential due to severe damage, a selective autophagic mechanism (mitophagy) mediated by the E3 ubiquitin ligase, Parkin, facilitates their turnover 2. Loss of function mutations in the Parkin gene (PARK2) are associated with early-onset forms of Parkinson’s disease (PD) suggesting a role in neuronal survival 3. Conversely, genetic enhancement of Parkin expression in the striatum increases dopaminergic neuron survival in rodent models of PD 4, which may occur through clearance of damaged mitochondrial DNA 5. Increasing PARK2 transcription is a compelling strategy for PD-relevant neuroprotection. Classically, expression levels have been pharmacologically modulated through two main mechanisms: distal regulation of the locus of interest via cell surface receptors that act through signaling pathways (e.g. MAPK), and proximal regulation through direct engagement of nuclear receptors 6. Recently, a refined understanding of heterochromatin dynamics has provided another access point by targeting epigenetic control of gene transcription 7. One approach to screen for compounds that increase gene expression uses bioluminescent enzyme sentinels such as luciferase to amplify subtle events over a broad response range 8. However, the resulting data is encumbered by enrichment of artifactual compound activities found in diverse compound libraries, irrespective of reporter type 9. Compounds that bind reporters, even weakly, increase reporter protein

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half-life and yield higher signal 9a, b, which is especially problematic for gain-of-signal assays. This limitation can be mitigated with a ‘coincidence reporter:’ a bicistronic transcript stoichiometrically translated into two non-homologous reporters by means of a highly efficient 2A ‘ribosomal skipping’ sequence (Fig. 1A, B). Use of orthogonal enzymology establishes a low probability that compounds will interact with both reporters, thus only ‘coincident’ responses reveal on-target activities 10. As illustrated in Figure 1A, a hypothetical transcriptionally active compound C1 will result in a concentration-dependent gain-of-signal response as measured by increases in both FLuc (green curve) and NLuc (blue curve) reporters. Further, if C1 is cytotoxic at higher tested concentration the concentration response curves (CRCs) will appear bell-shaped (solid lines), while no overt cell toxicity yields a sigmoidal CRC (dotted lines). However for the NLuc ligand C2 the CRCs for the FLuc and NLuc responses will be noncoincident, where increasing NLuc signal is caused by C2-based attenuation of NLuc protein turnover in the cell. As C2 exceeds its IC50 for NLuc (~equal to the apparent cell based EC50 for the ascending phase of the bell curve), C2 strongly inhibits the NLuc enzyme activity as measured during signal detection. Thus, the CRC profiles of the FLuc and NLuc signals can be used to discriminate between transcriptionally active compounds and artifacts that act by directly perturbing the basal reporter levels through a common ligand-based stabilization phenomenon. Additionally, by targeting coincidence reporter integration to a defined gene locus a further increase in biological fidelity can be achieved 11. Thus, combination of these approaches represents an advance in reporter cell line development for drug discovery and chemical biology.

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To capture responses over a variable concentration range, we miniaturized the assay into a 1536-well format and used titration-based screening, the basis of qHTS, to obtain dose-response curves for each compound in a chemogenomic library comprised of bioactive small molecules 12. This strategy identified chemotypes and putative pathways regulating PARK2, with a notable representation by epigenetic regulators, which were reinforced by functional genomics. We focused on inhibitors of cholesterol biosynthesis and the JNK family of MAP kinases, and provide preliminary data supporting a role for the latter in mitochondrial quality control. Simultaneously, we identified and removed from consideration reporter-biased false positives. The precision of these results establishes the gene locus-targeted coincidence reporter as a significant advancement in compound screening technology.

Results An assay to monitor endogenous PARK2 expression. To develop a model system compatible with a qHTS assay format (e.g. titrationbased screening in 1536-well plates), we sought a cell type that expresses detectable PARK2, can be expanded from single cells for clonal isolation, and is amenable to efficient reverse transfection to enable arrayed siRNA screening. BE(2)-M17 neuroblastoma cells meet these criteria and have proven useful for validating novel regulators of PINK1/Parkin-mediated mitochondrial quality control 13, 14. To evaluate endogenous PARK2 regulation, we utilized TALEN-mediated genome edited BE(2)-M17 cells to encode a coincidence reporter at the PARK2 locus (Fig. 1B). As an improvement over the proof-of-concept with Firefly (FLuc) and Renilla

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(RLuc) luciferases, we replaced RLuc with the recently developed NanoLuc (NLuc) 15, as NLuc luminescence is brighter and longer-lived. Fusion with a PEST sequence ensured that NLuc half-life was appropriately attenuated for a gain-of-signal cell-based assay 16. We isolated a clonal line (PTRC6: PARK2 Transcriptional Reporter Clone 6) containing the correct 5’ and 3’ integration junctions (Fig. S1A). PCR confirmed heteroallelic integration of the coincidence reporter, with retention of wild-type PARK2 allele(s) (Fig. S1B). During clonal screening, we tested basal and stimulated NLuc signal with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which is known to increase PARK2 expression 17 and observed >2-fold induction of NLuc signal (Fig. S1C). We further validated the PTRC6 assay by treating with CCCP or another known PARK2 enhancer, tunicamycin, which both increase FLuc signal (Fig. S1D). mRNA levels of reporter or wild-type PARK2 increased ~3-fold with CCCP, confirming transcript behavior corresponded to luciferase signals (Fig. 1C). To enable a qHTS format where both luciferase readouts could be acquired, we developed with Promega Corp. a prototype dual luciferase reagent (Nano-Glo DualLuciferase Reporter or Nano-Glo DLR). In this protocol FLuc is read first, and NLuc is read second with simultaneous quenching of the FLuc signal (see Table S1). Pilot screening found that the histone de-acetylase (HDAC) inhibitor panobinostat (LBH589) increased expression of the PARK2 locus more than CCCP. We therefore validated the assay with 500 nM panobinostat (Fig. 1D) and demonstrated signal and reproducibility (Z’ ≥ 0.5) for FLuc and NLuc (Fig. S1E) an excellent assay for HTS 18.

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Chemogenomic screening for PARK2 expression enhancers. We next developed a chemogenomic profile of bioactive compounds using 7-11 concentrations for pharmacological assessment (Table S2). We evaluated FLuc and NLuc responses using 4-parameter fits of the dose-response curves. Only compounds with high quality curve classes (1.1, 1.2, 2.1, 2.2; see SI Methods 12, were chosen as initial actives. Since biological actives should yield FLuc and NLuc responses with comparable potency, we used the concordance correlation coefficient (CCC) 19 to measure differences in response curves between channels. CCC analysis identified 19 non-coincident compounds from an initial list of 87 actives. Visual inspection of these indicated only 2 were actually non-coincident, producing a list of 85 sigmoidal actives (Fig. 2A; see Table S3). For bell-shaped dose-response curves, sigmoidal fitting fails to accurately assign curve class. To identify additional compounds with coincident responses, we re-analyzed the normalized data for values >3 SD higher than the median value at non-maximal concentrations in both channels, and applied the CCC filter to confirm that stimulation profiles were of comparable potency. This process identified 237 additional non-sigmoidal fit actives (Table S3). The selection strategy is outlined in Fig. S2A. Combining the coincidence strategy with a qHTS approach reduced the number of active compounds 58% over a hypothetical scenario of using FLuc alone or 62% for NLuc alone (Fig. 2A). Upon analyzing the interpolated ~10 µM data points for each compound, in the absence of titration-based screening, the coincidence reporter would have reduced the number of actives 77% using FLuc alone, and 72% using NLuc alone

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(Fig. S2B). These rates demonstrated a clear gain in efficiency over both single-point and pharmacological screening paradigms. We observed a variety of activity signatures across 7,456 compounds. Plotting the EC50 for each compound with activity in at least one channel, we obtained a correlation plot (Fig. 2B) that segregates true actives from potential artifactual responses (Table S3). We tested whether representative compounds with each activity signature were increasing PARK2 mRNA. Among compounds yielding an NLuc-specific response (Fig. 2C−D), a FLuc-specific response (Fig. 2E−G), and a coincident response (Fig. 2H) at ~EC90 concentrations, only the representative coincident active JQ-1 increased PARK2 mRNA (Fig. 2I). We next reacquired and assayed 209 actives in triplicate, including coincident and single-channel actives. The majority of compounds (128/160 coincident actives; 80%) reconfirmed from the primary screen (Table S3 and Fig. S2C), and exhibited similar sigmoidal (e.g. Fig. S2D) or non-sigmoidal (e.g., Fig. S2E) profiles.

Coincidence responses illuminate compound mechanism of action. In examining the structure-activity relationships (SAR) behind non-coincident NLuc or FLuc signatures, a series of dihydropyridines demonstrated exclusive increases in NLuc signal (Fig. S3A, Table S4). Among these, cilnidipine had no effect on PARK2 mRNA (Fig. 2D), further confirming coincidence reporter discrimination of an artifactual response 11. As dihydropyridine compounds modulate L-type calcium channel signaling 20

and some are approved for clinical use, they would otherwise be of interest for

modulating PARK2. To investigate these off-target activities on NLuc, we constructed a

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library of dihydropyridines and structurally unrelated L-type calcium channel blockers. We confirmed the NLuc-specific response with many of the dihydropyridine family members (Fig. 3A, Table S4). Interestingly, glibenclamide (Fig. 3B), which contains an aryl sulfonamide moiety known to stabilize RLuc, a related luciferase, exhibited an NLuc-specific response 10. None of the other structurally diverse L-type calcium channel agonists exhibited activity, indicating dihydropyridines induce an artifactual NLuc response. The new coincidence reporter using the NLuc-PEST reporter also detected proteasomal inhibitors (Fig. S3B, Table S4) through a distinct signature (FLuc, no response; NLuc > 250%). Prior work investigating compound stabilization of FLuc suggested that multiple chemotypes would yield FLuc-specific activity. Among chemotypes previously identified are the aniline aryloxazoles kinase inhibitors 21. Profiling two libraries of kinase inhibitors confirmed this activity, validating enzyme assay data (Fig. S4A and 21b, and identified a new cluster of aryloxazoles with FLuc-specific activity (Fig. S4B, Table S4). These examples highlight the gain in efficiency possible when reporter-biased artifacts can be eliminated during a primary screen.

Strong representation of epigenetic chemotypes among coincident actives. We next examined SAR relationships among highly coincident actives. Notably, the aryl azapine family of bromodomain inhibitors 22 drove potent coincident activity (Fig. 4A, Table S4). Cheminformatic clustering identified GYKI-53655 as belonging to this chemotype (Fig. 4A). As PTRC6 cells are brain-derived, we investigated GYKI53655 further. We confirmed a dose-dependent increase in PARK2 mRNA for two aryl

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azapines and GYKI-53655, and repeated the qHTS assay to verify compound behavior (Fig. 4B). Further analysis revealed that among the 25 AMPA receptor modulators screened only GYKI-53655 exhibited a coincident response (Table S4), suggesting GYKI-53655 acts through a unique mechanism independent of AMPA receptor modulation. Among coincident actives, we also noted another epigenetic-related enrichment of the hydroxamic acid motif characteristic of HDAC inhibitors (Fig. 4C, Table S4 23).

Pathway inference from chemogenomics and functional genomics. We examined the list of active compounds for known bioactivity that might provide insights about biological pathway modulation. In addition to epigenetic chemotypes such as the aryl azapines and hydroxamic acids, we observed activity from a number of protein kinase inhibitors (PKIs), the majority displaying low to modest potency. Given the nominal selectivity of most PKIs 24 we sought to align chemotype activity with data from a kinome-wide gene-silencing experiment to expose targets involved in PARK2 transcription. Using the PTRC6 assay we conducted a kinome-wide siRNA screen and selected genes with a p 150% in PARK2 expression over control, BRD4 uniquely yielded multiple actives, which is a strong indication of on-target activity 13. Therefore, it is likely that BRD4 negatively regulates the PARK2 locus. BRD4 inhibition acts as a surrogate for suppression of myc transcription factors, and despite traditionally being associated with increasing gene expression, N-myc can negatively regulate PARK2 expression 26 as has been reported for other targets 27. We confirmed that JQ-1 regulates N-myc degradation as reported 28, (Fig. S6A and quantified in Fig S6B), leading us to conclude that this is likely the mechanism of PARK2 regulation 26. We next investigated whether Parkin protein increased with treatment by different epigenetic modulators: the HDAC inhibitor panobinostat, the aryl azapine JQ-1, or the benzamide HDAC inhibitor MS-275, all of which evoked a strong response in both the PTRC6 coincidence reporter assay and qRT-PCR (Fig. 5B). A therapeutic approach directed toward the PARK2 locus should be accompanied by an increase in Parkin. Experimentally however, it has been observed that mRNA and corresponding protein levels do not necessarily change in synchrony 29. Thus the possibility exists that Parkin protein levels will not be coupled tightly to PARK2 transcript levels upon compound treatment due to mechanistically diverse compound behavior. We studied parental BE(2)-M17 cells, which express ~2-fold more Parkin than the PTRC6 cells, and in which maximal changes in protein are evident after 48 hours treatment. MS-275 increased Parkin protein, whereas JQ-1 and panobinostat had little effect (Fig. 5C), suggesting

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post-transcriptional mechanisms of these broad-acting compounds were counteracting the increase in PARK2 mRNA we observed by qRT-PCR (Figs. 2I and 4B). To explore the biological relevance of these compounds, we measured PARK2 mRNA in human iPSC-derived dopaminergic neurons. Both panobinostat and MS-275 significantly induce PARK2 mRNA in these cells (Fig. 5D). This suggests activity in dopaminergic neurons, but does not exclude the possibility that the response is due to a primary effect on non-dopaminergic cell types as this cell population is heterogeneous 13.

Cholesterol Biosynthesis regulates PARK2 Expression. Both chemogenomic and kinome siRNA data indicated the cholesterol biosynthesis pathway controls PARK2 expression (Fig. 5E). Our primary screen identified mevastatin, which inhibits HMG-CoA reductase, as increasing PARK2, and in follow-up analysis all 4 statins tested exhibited activity with varying potency (Fig. 5F and Fig. S5C). Validating this compound data, the second ranked active from the siRNA screen was PMVK (Table S5), which catalyzes the 5th step of the pathway. We acquired siRNA targeting PMVK and other pathway genes. PMVK knockdown phenocopied statin treatment, and knockdown of the upstream enzyme, MVK, likewise increased PARK2 reporter expression (Fig. S5D). siRNA knockdown of HMG-CoA reductase or synthase had little effect, perhaps due to incomplete knockdown (see Methods). To validate the effect observed in the primary screen, we tested the pro-drug mevastatin and the more potent of the mature drugs, fluvastatin, and found that both cause a dose-dependent increase in Parkin protein, with mevastatin toxicity reducing

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Parkin protein at the higher concentrations tested (Fig. 5G). These results support a previously unidentified effect of statins on PARK2.

JNK Inhibition increases Parkin and Mitochondrial Quality Control. We additionally sought compounds with reported neuroprotective function. Among the coincident actives were the inhibitors, SR-3306 and SP600125 (Table S3), described in the literature to target the JNK family of kinases 30. We focused on SR3306, the more potent of the inhibitors as demonstrated from our follow-up PTRC6 coincidence reporter confirmation data set (Fig. 6A, and highlighted in Fig. S2C). Similar to MS-275 or statin treatment, SR-3306 dose-dependently increases Parkin protein in BE(2)-M17 cells (Fig. 6B). Although kinase inhibitors such as SR3306 are unlikely to act selectively, we asked whether the reported neuroprotective function ascribed to SR-330631 may occur in part through increasing Parkin levels and mitochondrial quality control. To examine this we chose a model of mitochondrial damage caused by protein stress in the mitochondrial matrix driven by the doxycycline-induced expression of the misfolded ∆OTC protein 32. We observed that SR-3306 treatment partially cleared ∆OTC and increased Parkin (Fig. 6C−D). In contrast, MS-275, which robustly increases Parkin, did not induce ∆OTC clearance, likely due to pleiotropic effects, as HDAC inhibition is known to be non-selective. SR-3306 titration further made clear the correlation between ∆OTC clearance and increasing Parkin expression (Fig. S6F).

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Discussion This study describes the first successful implementation of the Firefly /NanoLuc Luciferase coincidence reporter as a qHTS-compatible assay, and identifies new pathways regulating PARK2. Reporter gene-specific responses mistakenly attributed to the action of compounds on true biology have confounded drug discovery since reporter genes were introduced over two decades ago 9b, 33. While the advantages of reporter genes for construction of HTS assays are clear 8, 34, only recently has an understanding of the major limitations emerged, and feasible solutions proposed 9d, 10. By combining developments in screening technologies with genome editing, we accelerated accurate identification of PARK2 locus-activating compounds. Titration based-screening, here performed as a pilot qHTS, allowed us to avoid false negatives arising from biphasic responses where the active concentration range is narrow. Such “bell-shaped” responses cannot be detected using single-point screening; one example is the kinase inhibitor SR-3306, which induced Parkin and mitochondrial quality control at low concentrations, but was toxic at higher concentrations. While qHTS is effective for eliminating false negatives, it does not identify false positives arising from reporter stabilization. To this end we used a coincidence reporter to differentiate NLuc- or FLuc-specific channel activity. Chemotypes eliminated from consideration, such as the dihydropyridines (NLuc-specific signal) or the aniline aryloxazoles (FLuc-specific signal), displayed SAR relationships that would have otherwise been investigated. The proportion of these compounds in large, diverse chemical libraries is reported to be from 1-10%, depending on the reporter 9d. Since compounds targeting poorly explored biological processes can be rare, their detection is

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easily overlooked in favor of higher potency actives displaying reporter-biased SAR 9d. Thus, the coincidence reporter will have even greater utility for discovery involving larger compound libraries. In screening a chemogenomic library we identified a significant number of compounds that are known to act through epigenetic mechanisms, a result partially attributable to direct integration of the reporter into the endogenous locus. Two predominant classes of epigenetic regulators identified were HDAC and bromodomain inhibitors. We demonstrated that HDAC inhibitors up-regulate PARK2 expression, an observation extended to iPS-derived dopaminergic neurons. Also noteworthy was our identification of a JQ-1 sensitive Brd4 circuit controlling PARK2 expression, identified through a combined chemogenomics and siRNA-mediated gene silencing approach. Genome editing of reporter genes into endogenous loci represents a major step forward in the design capability of physiologically relevant primary screening assays. Previous work using genome-integrated reporters from mouse models has allowed screening efforts directly in the cell type of interest 35. While using transformed cell lines can depart radically form physiological systems they offer a practical alternative in refining new techniques, as we show here, while retaining some pathways that may translate to more complex model systems. Currently, it is more technically feasible to perform genome editing in transformed cell lines as opposed to primary or pluripotent cells. We anticipate that futures advances will allow for the development of pluripotent, genome-edited cell lines which have both the scalability of cell lines combined with pathophysiological relevance of the desired cell type.

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Recent progress in the discovery and optimization of small molecule inhibitors for epigenetic processes illustrates the potential for this alternate avenue in regulating gene expression 7, 36. Many target proteins and pathways of the epigenome remain unidentified and poorly understood compared to more established targets such as nuclear-hormone receptors 37. Coupling genome-editing approaches to locus-targeted transcriptional-readout assays represents a promising means to enable discovery of phenotypically-defined epigenetic modulators. We also investigated compounds targeting other, potentially more circumscribed pathways. SR-3306, a putative JNK inhibitor previously demonstrated to have neuroprotective effects in rodents 38, cleared unfolded protein stress from mitochondria, which to our knowledge is the first description of such activity. These results suggest that the protective function of JNK inhibitors may occur in part through amplification of Parkin and mitochondrial quality control. The control of PARK2 expression by the cholesterol biosynthetic pathway represents an interesting convergence between our pharmacological and functional genomic data. PARK2 knockout rodents displayed altered lipid storage and metabolism 39

, and it is tempting to speculate that association between long-term statin

administration and lower rates of Parkinson’s disease 40 may involve an increase in Parkin. Brain penetrant statin-mediated reduction in inflammation might occur parallel with neuroprotection due to Parkin enhancement, and is an avenue to study the clinical relevance of Parkin modulation without novel compound development. The discovery of new Parkin pathway biology described here validates the power of the coincidence

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reporter approach, and opens new avenues for pursuit of PD therapeutics.

Experimental Procedures Assembly of recombination coincidence biocircuit cassette: Using genomic DNA purified from BE(2)-M17 cells (Zymo research catalog #D3024) as a template, an approximately one kb fragment of each the 5' homologous arm (5’HA) and the 3' homologous arm (3’HA) of DNA around the PARK2 start codon were PCR amplified with Phusion DNA polymerase (NEB). These fragments were then ligated together with the NanoLucPEST (NLucP)-polyA sequence PCR-amplified from pNL1.2 vector (Promega) into pBluescript SKII at EcoRI/BamHI sites to make Parkin-NLucP donor construct. This construct was then linearized by PCR and the FLuc-P2A fragment, which was PCRamplified from the pCI6.2 vector 10 was inserted in front of NLucP by the in-fusion HD cloning method (Clontech #638910) to assemble the finished Parkin-FLuc-P2A-NLucP donor cassette. All oligoes used in this study are listed in Table S1.

Data analysis and definition of the CCC score. The goal of the concordance correlation coefficient (CCC) is to quantify the degree to which the two concentration response curves move together or diverge across the concentrations tested

19

. Note that this

score does not require a curve fit and instead operates on the observed responses. For an point titration, the CCC score is defined

=

+

2 + ( 17

− )


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Equation 1 where we define

1

1 = ( − ) = , ;

1 = ( − )( − )

at the "’th concentration from the FLuc and NLuc channels respectively. The score ranges from -1 to 1, where a value of 1 indicates perfectly coincident curves (either both decreasing or both increasing). A value of -1 will occur when the two curves are equal but opposite. As the concordance between the two curves, going in the same direction, increases, the score becomes increasingly positive.

Additional experimental procedures can be found in the Supplemental Information.

Author Contributions: SH, AF, RJY, and JI designed experiments, analyzed the results, and wrote the manuscript. CW engineered the coincidence reporter constructs and cell line. SH, AF and SM performed siRNA experiments. SH, AF, RM, RG, and JI performed 18



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cheminformatics analysis. RM and RG developed the methodology for scoring coincidence. SH and SHA performed experiments in differentiated iPSCs. SH optimized the assay and performed primary screening and qPCR. AF performed follow-up screening and secondary assays involving immunoblotting.

Acknowledgements- We thank C. Eggers, B. Binkowski, and K. Kopish of Promega for development of the NanoDLR assay reagent used in this study, P. Dranchak and the members of the NCATS engineering core for assistance with liquid handling systems, M. Rao’s lab at NIH for assistance with iPS neuron cultures, and D. Leja for graphic abstract artwork. This work was supported by the NCATS (J.I.) and NINDS (R.J.Y.) intramural program and The Michael J. Fox Foundation (grant 8966 awarded to J. Inglese and R. J. Youle).

References

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Figures and Legends

Figure 1: Development of a coincidence reporter qHTS assay for endogenous PARK2 expression. (A) Overview of coincidence reporter (CR) concept for chemical library HTS. Combined outputs from individual reporters encode mechanism of compound activity as shown conceptually in model concentration response plots. For example, compound C1 acts on a target within a cellular pathway, as reporters respond similarly in an unbiased manner, while the non-coincident response of C2 indicates stabilization of the NLuc reporter. (B) Graphical representation of TALEN-mediated genome editing strategy for coincidence reporter insertion into the PARK2 locus. In a BE(2)-M17 neuroblastoma cell line, a TALEN pair was used to generate double-strand DNA breaks (site in red near the ATG start codon) and stimulate homologous recombination of sequence containing the FLuc-P2A-NLuc-PEST coincidence reporter from a HDR vector. All primers used to create the HDR vector are listed in Table S1. 23


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(C) qRT-PCR analysis of mRNA from PTRC6 cells after treatment with 10 µM CCCP or DMSO for 24 hours. White bars represent levels of the coincidence reporter mRNA, and grey bars represent levels of the wild-type PARK2 mRNA. (D) Bioluminescent output from assay validation plates (sample image in Fig. S1E) demonstrate assay performance in FLuc (green; left y-axis) and NLuc (blue; right y-axis) channel. Data shown for three replicate plates, left bar graph in FLuc or NLuc represents DMSO control (hatched) and treatment with 500 nM panobinostat (solid). Bars in (C) and (D) represent mean ±S.D. of 3 independent experiments.

Figure 2: Activity signatures from chemical library qHTS. (A) Distribution of coincident and non-coincident activity as determined by 4-parameter curve fitting and qHTS curve classification assignments and CCC analysis. qHTS performance metrics are given in Table S1. Total Coincident Actives is the sum of sigmoidal and non-

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sigmoidal fit actives; inconclusive and single channel actives are binned separately (Table S3). (B) Correlation plot of pEC50 values for NLuc and FLuc for all compounds exhibiting activity primary qHTS screening of PTRC6 cell line. Compounds with activity in only the FLuc channel are binned by number in green on y-axis; light-green represents 5-parameter fit FLuc only responses. Compounds with activity in only the NLuc channel are binned by number in blue on x-axis; light-blue represents 5-parameter fit NLuc only responses. Compounds with activity in both channels are distinguished by pharmacology: open circles correspond to a 4-parameter sigmoidal response, and open squares correspond to a 5-parameter non-sigmoidal response curves. Concentration response curves for compounds displaying non-coincident NLuc (C−D), non-coincident FLuc (E−G), or coincident FLuc and NLuc signal (H). Graphs display activity normalized to neutral and positive controls for FLuc curve (green) and NLuc curve (blue). CCC: concordance correlation coefficient, a parameter that measures concordance of the NLuc and FLuc curves, with 1 representing perfect concordance. (I) Measurement of PARK2 mRNA by qPCR after treatment with compounds as in (C−H). ns; nonsignificant, ***; (P

Chemogenomic Profiling of Endogenous PARK2 Expression Using

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