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Published in final edited form as: Cell Rep. 2015 Mar 26;11(1):43–50. doi: 10.1016/j.celrep.2015.03.001

An In vivo Chemical Genetic Screen Identifies Phosphodiesterase 4 as a Pharmacological Target for Hedgehog signaling Inhibition

Charles H Williams 1,2,3,#, Jonathan E Hempel 1,2,#, Jijun Hao 1,8, Audrey Y Frist 1, Michelle M Williams 1, Jonathan T Fleming 3, Gary A Sulikowski 2,6, Michael K Cooper 5,7, Chin Chiang 3, Charles C Hong 1,2,3,4,7,§
PMCID: PMC4394042  NIHMSID: NIHMS669677  PMID: 25818300

SUMMARY

Hedgehog (Hh) signaling plays an integral role in vertebrate development, and its dysregulation has been widely accepted as a driver of numerous malignancies. While a variety of small molecules target Smoothened (Smo) as a strategy for Hh inhibition, Smo gain of function mutations have limited their clinical implementation. Modulation of targets downstream of Smo could define a paradigm for treatment of Hh-dependent cancers. Here, we describe eggmanone, a small molecule identified from a chemical genetic zebrafish screen which induced a Hh-null phenotype. Eggmanone exerts its Hh-inhibitory effects through selective antagonism of phosphodiesterase (PDE) 4, leading to protein kinase A activation and subsequent Hh blockade. Our study implicates PDE4 as a target for Hh inhibition, suggests an improved strategy for Hh-dependent cancer therapy and identifies a unique probe of downstream-of-Smo Hh modulation.

INTRODUCTION

Hedgehog (Hh) signaling represents an important therapeutic target for the treatment of cancer. While its signal transduction and resulting downstream gene transcription are essential to vertebrate embryonic patterning and development, aberrant Hh signaling is responsible for a variety of malignancies including basal cell carcinoma (BCC), medulloblastoma, small cell lung cancer, and pancreatic cancer (Kar et al., 2012; Ng and Curran, 2011). Classical Hh signaling requires the presence of the extracellular ligand Sonic hedgehog (Shh), which upon binding to the transmembrane receptor Patched (Ptc) causes Ptc to remove its inhibitory influence on the G protein-coupled receptor Smoothened (Smo) (Ryan and Chiang, 2012). Activation of Smo then leads to nuclear translocation of the Gli family of transcription factors and induction of Hh target gene transcription. In the absence of the hedgehog family of ligands, Gli-mediated transcription is inhibited, and although numerous components of signal transduction between Smo activation and Gli-mediated gene transcription have been identified, their mechanism of action is not fully understood.

A wide array of small molecules that target Smo, including the canonical Hh inhibitor cyclopamine, have been shown to affect tumor progression (Taipale et. al., 2000; Carney and Ingham, 2013). Additionally, the Smo antagonist vismodegib is approved by the United States Food and Drug Administration (FDA) for the treatment for advanced BCC (Robarge et al., 2009; Von Hoff et al., 2009). However, oncogenic mutations downstream of Smo and acquired resistance due to Smo binding pocket mutations have limited the efficacy of this and other clinically promising therapeutics (Yauch et al., 2009). Therefore, the identification of distal signaling mediators could represent new therapeutic opportunities for Hh-dependent malignancies.

RESULTS

Discovery of eggmanone, a small molecule hedgehog inhibitor, from an in vivo chemical genetic screen

A screen of approximately 30,000 small molecules for their ability to effect alterations in embryonic zebrafish patterning identified a series of structurally related compounds represented by the prototype termed eggmanone (Figures 1A, S1A–S1C, Supplemental Experimental Procedures). Eggmanone reliably and selectively reproduced the zebrafish Hh-null phenotype with features ranging from ventral tail curvature, small eyes, loss of pectoral fins and enlarged, rounded somites to loss of neurocranial chondrogenesis and impaired slow muscle formation (Figure 1B–1D) (Barresi et al., 2000; van Eeden et al., 1996a; van Eeden et al., 1996b; Wada et al., 2005; Hirsinger et al., 2004). Consistent with loss of Hh signaling, we confirmed abrogation of the Hh-target gene Patched (Ptc)-1 expression in bud-stage adaxial cells, pectoral fin fields, and somites resulting from eggmanone treatment (Figure 1E, 1F).

Figure 1. Eggmanone affects embryonic zebrafish patterning through inhibition of Hedgehog signaling.

Figure 1

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(A) Chemical structure of eggmanone (Egm), (3-(2-methylallyl)-2-((2-oxo-2-(thiophen-2-yl)ethyl)thio)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-4(3H)-one). (B) Zebrafish embryos treated with 2 μM Egm starting at 4-hours post fertilization (hpf) exhibited a range of phenotypes found in Hh pathway mutants, including ventral tail curvature, loss of pectoral fins, and smaller eyes. (C) Alcian blue staining following 2 μM Egm treatment at 4-hpf revealed altered craniofacial development including the jaw. (D) Trunk slow muscles immunostained with anti-MyHC antibody (F59) showed altered slow muscle formation upon Egm treatment (2 μM). (E) Egm treatment (1 μM) abolished Hh-responsive Ptc1 expression in adaxial cells at 12 hpf (arrows). (F) Egm treatment (1 μM) ablated Hh-responsive Ptc1 expression in the pectoral fin bud at 48-hpf (arrows and asterisks). (G) Egm inhibited Sonic hedgehog (Shh)-responsive Gli-luciferase (Gli-Luc) reporter activity in a dose-dependent manner when stimulated with Shh conditioned medium (n = 4 for each condition, results represented as mean relative luciferase units (RLU) ± standard error of the mean (SEM); p-value <0.0184, starting at 1 μM). (H) Egm inhibited purmorphamine-induced (3 μM) Gli-Luc reporter activity in a dose-dependent manner (mean ± SEM, n = 4 for each condition; p-value <0.0054, starting at 0.5 μM). (I) Egm inhibited purmorphamine-induced (3 μM) Ptc1 expression in NIH3T3 fibroblasts (mean ± SEM, n = 3, expression normalized to GAPDH, p-value <0.003, starting at 1 μM). Related to Figures S1 and S3.

Given that eggmanone elicited a loss-of-Hh phenotype in zebrafish, we tested its ability to directly affect Hh signaling in the mouse Hh reporter cell line Shh-Light2 (Taipale et. al., 2000). Eggmanone inhibited Hh-inducible Gli-responsive luciferase (Gli-Luc) activity in a dose-dependent manner when stimulated with the exogenous ligand Shh (Figure 1G). In contrast, cells transiently overexpressing Gli2 retained Gli-Luc activity following eggmanone treatment (Figure S1D). Moreover, developmental signaling pathways such as BMP (Bone Morphogenic Protein) were not perturbed by eggmanone (Figure S1E). Taken together, our results indicate that eggmanone pharmacologically targets an evolutionarily conserved component specifically in the Hh pathway upstream of Gli.

Eggmanone exerts its Hh-inhibitory effects downstream of Smo

Considering all new molecular entities registered with the FDA to date for Hh-driven clinical trials target the cyclopamine binding site of Smo, we examined eggmanone’s effects at the level of Smo signaling (Trinh et al., 2014). Specifically, eggmanone retained its Hh-inhibitory activity when Shh-Light2 cells were stimulated with the Smo agonist purmorphamine, blocking both Gli-Luc activity and Ptc1 expression (Figure 1H, 1I) (Sinha and Chen, 2005). Importantly, eggmanone failed to compete for Smo binding with BODIPY-cyclopamine when HEK293 cells overexpressing Smo were pre-treated with 5 nM of the fluorescent ligand followed by treatment with concentrations of eggmanone up to 10 μM (Figure S1F) (Chen et al., 2002a). Therefore, these results confirmed that eggmanone targets the Hh pathway at or downstream of Smo.

To rule out its action at alternative Smo binding sites, we investigated eggmanone’s ability to inhibit Hh signaling in Sufu−/− mouse embryonic fibroblasts (MEFs), which display constitutively active signaling downstream of Ptc and Smo (Chen et al., 2009; Lin et al., 2014). Specifically, eggmanone significantly reduced transcription levels of Gli1 and Ptc1, whereas, as expected, the Smo antagonist cyclopamine showed no inhibition (Figure 2A). Thus, taken in concert with our previous findings, eggmanone acts between Smo and Gli-mediated transcription to block Hh signaling.

Figure 2. Eggmanone alters the activity of Gli transcription factors.

Figure 2

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(A) Quantitation of mRNA transcripts of Gli1 and Ptc1 in Sufu−/− cells when treated with 10 μM Cyc (mean ± SEM, n = 7, not significant) or 10 μM Egm (mean ± SEM, n = 10, *** = <0.0001), expression normalized to GAPDH. (B) Immunostaining for the cilium marker Arl13b (red) and Gli2 (green) of unstimulated MEFs (top), MEFs stimulated with SAG (20 nM) in the presence of DMSO control (middle) or 5 μM Egm (bottom). Co-localization of Gli2 (yellow) in the primary cilium remained unchanged in Egm treated MEFs. Representative cilium (white box) magnified to the right, scale bar of left column 10 μm; columns 2–4 scale bar 0.2 μm. (C) Representative western blot for full length Gli2 in nuclear (top, n = 4) and whole cell (middle, n = 2) fractions of NIH3T3 cells. Western blot of Gli3 (bottom, n = 3) from whole cell lysate of C3H10T1/2 cells. Neg, unstimulated. SAG, stimulated with SAG (100 nM) for 1.5 hours (Gli2) or 24 hours (Gli3). SAG+FSK, co-treated with SAG and FSK (30 μM). SAG+Egm, co-treated with SAG and Egm (10 μM). Corresponding western blots for nuclear Lamin-A/C and whole cell α-tubulin as loading controls. FL, full-length, active forms of Gli2 and Gli3. R, repressor form of Gli3. (D) Quantitative analysis of the mean ratio of normalized nuclear full length Gli2 to normalized whole cell Gli2 from (C). (E) Quantitative analysis of the ratio of full length to repressor form of Gli3 from (C) (mean ± SEM, n = 3 for each condition; ** = 0.0095; * = 0.028, vs. SAG). Related to Figure S2.

Eggmanone differentially affects Gli transcription factors

The primary cilium plays a critical role in Hh signal transduction (Corbit et al., 2005; Rohatgi et al., 2007). Smo translocation to the cilium propogates Hh pathway activation through the ciliary trafficking of Gli (Wong et al., 2009). Therefore, we examined whether eggmanone affected Gli trafficking to the primary cilium, where Gli transcription factors undergo activation prior to proteolytic processing and subsequent translocation to the nucleus to either promote or inhibit transcription (Haycraft et al., 2005; Liu et al., 2005). Thus, under pathway stimulation with SAG (Frank-Kamenetsky et al., 2002; Chen et al., 2002b), a Smo agonist, MEFs stained for Gli2 confirmed trafficking of Gli2 to the primary cilium (Figure 2B). Importantly, with co-treatment of SAG and eggmanone, Gli2 properly localized to the proximal tip of the cilium as marked by the ciliary maintenance protein Arl13b. Additionally, with the recent report of ciliobrevin D, a small molecule that antagonizes the cytoplasmic motor dynein resulting in defective retrograde transport and causes gross ciliary malformation, we sought to examine whether eggmanone affected cilium structure and trafficking (Hyman et al., 2009; Firestone et al., 2012). In NIH3T3 cells stained for the ciliary maintenance protein Arl13b, cilium structure was unaffected by eggmanone, and in contrast to ciliobrevin D, the ciliary transport protein IFT88 remained intact following eggmanone treatment (Figure S2). Thus, in light of unaltered structure and ciliary trafficking of Gli, we focused on elucidating eggmanone’s effects on Gli processing and nuclear translocation.

Under regulation by Hh signaling, each Gli transcription factor is differentially processed to form either Gli full length (FL) activators or Gli repressors (R) which then translocate to the nucleus to either activate or inhibit downstream gene transcription. However, the relative importance of Gli processing and nuclear translocation varies between individual factors. For instance, the differential processing of Gli3 is more sensitive to the Hh pathway status than Gli2, which remains predominantly in its GliFL form regardless of Hh activity (Hui and Angers, 2011). Thus, we separately investigated Gli2FL nuclear accumulation and proteolytic processing of Gli3FL to Gli3R. Hh pathway stimulation with SAG led to an increase in the fraction of Gli2FL in the nucleus, and this nuclear accumulation was inhibited by eggmanone and the adenylyl cyclase (AC) activator forskolin (FSK) (Figures 2C, 2D). SAG treatment also inhibited processing of Gli3FL to Gli3R, and both eggmanone and FSK restored Gli3R formation (Figures 2C, 2E) (Humke et al., 2010). Taken together, these data suggest that eggmanone functions upstream of Gli processing and nuclear translocation to exert its Hh-inhibitory effects.

Eggmanone causes selective activation of protein kinase A at the basal body

An emerging understanding of Gli transcription factor activity involves direct phosphorylation by PKA at two separable steps: repressor formation, and activation for nuclear translocation (Pan et al., 2009; Wolff et al., 2013; Tuson et al., 2011; Wang et al., 1999; Zeng et al., 2010; Niewiadomski et al., 2014). As our Gli2 and Gli3 western data (Figure 2) correlated well with this picture, we next investigated eggmanone’s effects on PKA autophosphorylation. Immunostaining and quantification of phospho-threonine 197-PKA (p-T197-PKA) following eggmanone treatment strikingly indicated a significant increase in PKA activity at the base of the primary cilium, corresponding to the basal body, when co-stained with the ciliary maintenance protein Arl13b (Figures 3A, 3B). However, western blotting of whole cell lysates indicated no significant increase in p-PKA levels upon eggmanone treatment (Figure 3C), hinting at the possibility of localized PKA activation by eggmanone. PKA’s localization to the basal body has been previously reported as a mechanism for control of Gli processing prior to its trafficking to the nucleus to inhibit Hh signaling (Tuson et al, 2011). To confirm this localization, co-staining of p-T197-PKA and γ-tubulin, which associates with the basal body, further revealed robust PKA activation at the basal body due to eggmanone treatment (Figure 3D, 3E). Taken together with the Gli processing and translocation data, localized PKA activation may be responsible for mediating eggmanone’s effects on Hh signaling.

Figure 3. Eggmanone modulates the activity of PKA at the basal body.

Figure 3

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(A) Immunostaining for the cilia marker Arl13b (green) and the autophosphorylated form of the PKA catalytic subunit (phospho-T197-PKA; red) in NIH3T3 cells unstimulated (left), or stimulated with the Smo agonist SAG (20 nM, middle) demonstrated a low baseline PKA activation; co-treatment with SAG and Egm (5 μM, right) increased local PKA activation at the base of the primary cilium. Representative cilium (white box) magnified in the inset. Scale bars 10 μm. (B) Quantitative analysis of the local activation of phospho-T197-PKA at the cilium (C) and peri-cilium (P) (mean ± SEM, n = 3 for each condition, * = 0.031 vs. SAG alone at the peri-cilium), as shown in (A). (C) Western blot of NIH3T3 whole cell lysates for phospho-T197-PKA (bottom) and α-tubulin (top). SAG was treated at 20 nM and Egm at 5 μM. (D) Immunostaining for the basal body marker γ-tubulin (green) and the autophosphorylated PKA catalytic subunit (phospho-T197-PKA; red) in NIH3T3 cells unstimulated (top), or stimulated with SAG (20 nM, middle) and Egm (5 μM) (bottom). Scale bars 10 μm. (E) Quantitative analysis of p-PKA intensity at the basal body from (D); RFU, relative fluorescence units (mean ± SEM, n = 21 for each condition, *** = <0.0001 vs. SAG alone).

Eggmanone inhibits phosphodiesterase 4

PKA is anchored to the basal body by A kinase-anchoring proteins (AKAPs) and is activated in response to the ubiquitous secondary messenger cyclic adenosine monophosphate (cAMP) (Terrin et al, 2012). Cyclic AMP is synthesized by ACs, but most importantly, its levels are controlled through its degradation by phosphodiesterases (PDE). Following this logic, we hypothesized that eggmanone may antagonize Hh signaling by PDE inhibition. In a screen of PDE super-family members, eggmanone potently inhibited PDE4D3 and the activity of three other members by 50% or greater at 20 μM (Figure 4A). In a dose-response assay with these four classes of PDEs, eggmanone displayed potent antagonism of PDE4D3 with an IC50 of 0.072 μM, approximately 40- to 50-fold selective over other PDEs (Figure 4B). Lending further validity to PDE4 as eggmanone’s cellular target, PDE4D3 has been shown to complex with PKA and AKAP9 at the centrosome (Terrin et al., 2012). Additionally, a counter-screen of eggmanone against a broad and comprehensive panel of 442 kinases, 158 GPCRs, and 21 phosphatases revealed that no other targets were inhibited by eggmanone by greater than 50% (Tables S1, S2, and S3). Based on these results, we conclude that eggmanone is a potent and selective PDE4 inhibitor.

Figure 4. Eggmanone exerts its Hedgehog-inhibitory effects through antagonism of phosphodiesterase 4.

Figure 4

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(A) In vitro PDE activity assays across ten PDE super-family members by Egm (20 μM). (B) Dose response curve of Egm against PDE4D3 (red), PDE3A (blue), PDE10A2 (purple), PDE11A4 (green). IC50s were 0.072 μM, 3.00 μM, 3.05 μM and 4.08 μM respectively. (C) Transient overexpression of wild type PDE4D3 induced Hh reporter activity (* = 0.0026 vs. pCS2 control), which was abolished by 5 μM Egm (p <0.0001 vs. PDE4D3 WT). Transient overexpression of dominant negative (DN) PDE4D3 decreased Hh reporter activity (* = 0.0121 vs. pCS2 control). Data is reported at mean ± SEM. (D) Rolipram (Rol) inhibited Sonic hedgehog (Shh)-responsive Gli-luciferase (Gli-Luc) reporter activity when stimulated with Shh conditioned medium (n = 4 for each condition, results represented as mean RLU ± SEM, p-value <0.003, starting at 1 μM). Related to Tables S1, S2, and S3.

PDE4 modulates Hh signaling in vitro

Considering the previous lack of direct correlation between PDE inhibition and Hh blockade, we sought to molecularly confirm this association. First, the PDE isoform most potently inhibited by eggmanone, PDE4D3, was transfected into Shh-Light2 cells and significantly increased Hh signaling (Figure 4C). This increased signaling was returned to basal level upon treatment with eggmanone. Furthermore, transfection of a dominant negative catalytically inactive PDE4D3 construct led to a reduction in Hh signaling which was further abrogated by subsequent eggmanone treatment (Figure 4C). Finally, if PDE inhibition modulates Hh signaling, we reasoned that the broad-spectrum PDE4 inhibitor rolipram should display inhibitory activity in the Gli-Luc reporter assay. Indeed, Shh-Light2 cells stimulated with Shh followed by treatment with increasing concentrations of rolipram did reduce Hh signaling levels, albeit with lower potency than eggmanone (Figure 4D). These results confirm that decreased cAMP concentrations due to higher PDE4 activity can promote Hh signaling, and the converse situation can lead to Hh inhibition.

DISCUSSION

Hh and PDE4 have been independently identified as promising targets for cancer therapy. As previously mentioned, Hh signaling has been shown to drive tumor progression in BCC, medulloblastoma, and other cancers (Kar et al., 2012; Ng and Curran, 2011). PDE4 has primarily been implicated as a driver of central nervous system (CNS) tumors such as medulloblastoma and glioblastoma as well as lung and breast tumors (Goldhoff et al., 2008; Sengupta et al., 2011). Moreover, much evidence points to overexpression of PDE4 in a wide variety of tumors, and the resulting decrease in cAMP has been associated with increased prevalence of malignancies. Finally, recent studies have lent credence to PDE inhibition as relevant for Hh-driven cancers but have yet to elucidate the precise mechanism of action (He et al., 2014; Powers et al., 2015).

Our results demonstrate that eggmanone functions as a PDE4 inhibitor to inhibit Hh signaling downstream of Smo, the singular target of the currently FDA-approved Hh inhibitor vismodegib as well as Hh inhibitors being evaluated in clinical trials. Additionally, our data corroborate earlier studies that PKA plays an essential yet incompletely understood role in the regulation of Hh signals. Earlier studies found that the global PKA activator forskolin (FSK) abrogated Gli translocation to the primary cilium, in contrast to eggmanone, which does not disrupt Gli2 ciliary trafficking (Tukachinsky et al., 2010). We attribute this and other differences between eggmanone and FSK to the possibility that FSK’s effect on Gli ciliary trafficking may be unrelated to PKA activation. For instance, FSK blocks Gli2 ciliary localization in PKA−/− cells (Tuson et al., 2011) and inhibits translocation of phosphomimetic forms of Gli2 to the cilium even though the status of Gli phosphorylation by PKA does not affect Gli ciliary localization (Zeng et al., 2010). Ultimately, our results indicate that PKA activation by eggmanone disrupts a process downstream of Gli ciliary trafficking, which is further supported by our finding that eggmanone can inhibit Hh signaling downstream of Sufu, a key regulator of Gli distribution.

While the cellular effects of PKA have traditionally been viewed in the context of global signaling, recent studies highlight the importance of PKA localization in Hh regulation (Barzi et al., 2010; Niewiadomski et al., 2013). Consistent with the latter view, our study with eggmanone suggests that PKA activation at the basal body functions to regulate Gli processing and translocation after transit through the primary cilium, where PDE4 resides in the basal body complex to control local levels of cAMP and thus PKA activity. Thus, in contrast to FSK, eggmanone can be considered as a unique probe of localized PKA signaling, and our studies lend further validity to the unbiased in vivo chemical genetic screen for identifying valuable chemical biology tools. Importantly, we have further validated the concept of PDE4 as an important therapeutic target for Hh-driven cancers downstream of Smo and suggest a mechanism for PDE4 regulation of Hh signals.

EXPERIMENTAL PROCEDURES

All zebrafish experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee.

Chemical Screen

Chemical screening for small molecules that perturb zebrafish embryonic development was performed as previously described (Hao et al., 2010; Hong 2009) and is detailed in the Supplemental Information.

Eggmanone Synthesis

Methods for synthesis of eggmanone are detailed in the Supplemental Information and the synthetic scheme is represented in Figure S3.

Whole-Mount Zebrafish In-Situ Hybridization

In situ hybridization was performed as previously described (Westerfield 2000). Zebrafish Ptc1 probes were produced as previously described (Concordet et al., 1996).

Immunocytochemistry

NIH3T3 cells seeded on coated coverslips were treated with SAG (20 nM) in the presence or absence of Egm (5 μM). Cells with no SAG treatment served as a control. Cells were fixed, permeabilized, and stained for appropriate proteins. A more detailed protocol with antibody descriptions is detailed in the Supplemental Information.

Protein Isolation and Western Blotting

Unstimulated cells or cells treated with SAG (100 nM) in the presence or absence of Egm (10 μM) or FSK (30 μM) were lysed to obtain total cellular protein or fractionated for nuclear/cytoplasmic proteins. After separation and transfer, blots were incubated with primary then secondary antibodies. Blots were visualized using a LI-COR Odyssey system. A more detailed protocol with antibody descriptions is detailed in Supplemental Information.

Supplementary Material

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Acknowledgments

We thank Miles Houslay (University of Edinburgh) for the PDE4D3 construct and Tamara Caspary (Emory University) for the Arl13b antibody. We also thank Jonathan Eggenschwiler (University of Georgia) for the Gli2 antibody. Eggmanone was synthesized by the VICB Chemical Synthesis Core. JEH is supported by an American Heart Association Postdoctoral Fellowship (14POST19550002), and CCH is supported by a Veterans Affairs Career Development Transition Award, VA Merit Award 01BX000771, the Developmental Grants from the Center for Research in Fibrodysplasia Ossificans Progressiva and Related Disorders, and NIH R01HL104040.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures, five figures and three tables.

AUTHOR CONTRIBUTIONS

C.H.W., J.E.H., J.H., and C.C.H. designed experiments. C.H.W., J.E.H., J.H., A.Y.F., M.M.W., and J.T.F. performed experiments. C.H.W., J.E.H., J.H., A.Y.F., M.K.C., C.C., and C.C.H. analyzed data. G.A.S. provided support for synthesis of eggmanone. C.H.W., J.E.H., and C.C.H. wrote the manuscript with input from co-authors. M.K.C. and C.C. provided valuable manuscript feedback.

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Supplementary Materials

1
NIHMS669677-supplement-1.docx (88.9KB, docx)
2
NIHMS669677-supplement-2.pdf (1.2MB, pdf)
3
NIHMS669677-supplement-3.xls (65.5KB, xls)
4
NIHMS669677-supplement-4.xls (36KB, xls)
5
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