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A cell-based bioluminescence reporter assay of human Sonic Hedgehog protein autoprocessing to identify inhibitors and activators

Open AccessPublished:November 15, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102705

      ABSTRACT

      The Sonic hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and covalently attach cholesterol to the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor’s C-terminal enzymatic domain. A method to screen for small molecule regulators of this process may be of therapeutic value. Here we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing non-invasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing using endogenous cholesterol to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a wild-type (WT) SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. Additionally, a conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the activation of the cholesterol substrate. Inducibility of the D46A reporter was established using a synthetic sterol, 2-α carboxy cholestanol, designed to bypass the defect through intra-molecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel anionic phosphonylated coelenterazine, CLZ-P, as the nanoluciferase substrate. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated.

      Graphical abstract

      INTRODUCTION

      Sonic Hedgehog (SHh) ligands initiate vital cell signaling necessary for brain development and neural stem cell proliferation, while displaying oncogenic activity when dysregulated in sporadic tumors.
      • Rallis A.
      • Navarro J.A.
      • Rass M.
      • Hu A.
      • Birman S.
      • Schneuwly S.
      • Therond P.P.
      Hedgehog Signaling Modulates Glial Proteostasis and Lifespan.
      • Lai K.
      • Kaspar B.K.
      • Gage F.H.
      • Schaffer D.V.
      Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo.
      • Liu Z.
      • Xu J.
      • He J.
      • Zheng Y.
      • Li H.
      • Lu Y.
      • Qian J.
      • Lin P.
      • Weber D.M.
      • Yang J.
      • Yi Q.
      A critical role of autocrine sonic hedgehog signaling in human CD138+ myeloma cell survival and drug resistance.
      • Bissey P.A.
      • Mathot P.
      • Guix C.
      • Jasmin M.
      • Goddard I.
      • Costechareyre C.
      • Gadot N.
      • Delcros J.G.
      • Mali S.M.
      • Fasan R.
      • Arrigo A.P.
      • Dante R.
      • Ichim G.
      • Mehlen P.
      • Fombonne J.
      Blocking SHH/Patched Interaction Triggers Tumor Growth Inhibition through Patched-Induced Apoptosis.
      • Hong S.
      • Hu P.
      • Jang J.H.
      • Carrington B.
      • Sood R.
      • Berger S.I.
      • Roessler E.
      • Muenke M.
      Functional analysis of Sonic Hedgehog variants associated with holoprosencephaly in humans using a CRISPR/Cas9 zebrafish model.
      • Roessler E.
      • El-Jaick K.B.
      • Dubourg C.
      • Velez J.I.
      • Solomon B.D.
      • Pineda-Alvarez D.E.
      • Lacbawan F.
      • Zhou N.
      • Ouspenskaia M.
      • Paulussen A.
      • Smeets H.J.
      • Hehr U.
      • Bendavid C.
      • Bale S.
      • Odent S.
      • David V.
      • Muenke M.
      The mutational spectrum of holoprosencephaly-associated changes within the SHH gene in humans predicts loss-of-function through either key structural alterations of the ligand or its altered synthesis.
      • Chen M.
      • Tanner M.
      • Levine A.C.
      • Levina E.
      • Ohouo P.
      • Buttyan R.
      Androgenic regulation of hedgehog signaling pathway components in prostate cancer cells.
      The role of SHh signaling in health and disease, particularly in cancer, has prompted efforts to identify clinically useful antagonists of different pathway components.
      • Quaglio D.
      • Infante P.
      • Di Marcotullio L.
      • Botta B.
      • Mori M.
      Hedgehog signaling pathway inhibitors: an updated patent review (2015-present).
      • Jamieson C.
      • Martinelli G.
      • Papayannidis C.
      • Cortes J.E.
      Hedgehog Pathway Inhibitors: A New Therapeutic Class for the Treatment of Acute Myeloid Leukemia.
      • Petrova E.
      • Rios-Esteves J.
      • Ouerfelli O.
      • Glickman J.F.
      • Resh M.D.
      Inhibitors of Hedgehog acyltransferase block Sonic Hedgehog signaling.
      • Lauth M.
      • Bergstrom A.
      • Shimokawa T.
      • Toftgard R.
      Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists.
      • Rodgers U.R.
      • Lanyon-Hogg T.
      • Masumoto N.
      • Ritzefeld M.
      • Burke R.
      • Blagg J.
      • Magee A.I.
      • Tate E.W.
      Characterization of Hedgehog Acyltransferase Inhibitors Identifies a Small Molecule Probe for Hedgehog Signaling by Cancer Cells.
      Many SHh signaling inhibitors target Smoothened (SMO), a cell surface receptor involved in SHh signal transduction
      • Quaglio D.
      • Infante P.
      • Di Marcotullio L.
      • Botta B.
      • Mori M.
      Hedgehog signaling pathway inhibitors: an updated patent review (2015-present).
      ; however mutational escape from the anti-tumor effects of those SMO inhibitors presents a challenge to durable remission.
      • Yauch R.L.
      • Dijkgraaf G.J.
      • Alicke B.
      • Januario T.
      • Ahn C.P.
      • Holcomb T.
      • Pujara K.
      • Stinson J.
      • Callahan C.A.
      • Tang T.
      • Bazan J.F.
      • Kan Z.
      • Seshagiri S.
      • Hann C.L.
      • Gould S.E.
      • Low J.A.
      • Rudin C.M.
      • de Sauvage F.J.
      Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma.
      • Dijkgraaf G.J.
      • Alicke B.
      • Weinmann L.
      • Januario T.
      • West K.
      • Modrusan Z.
      • Burdick D.
      • Goldsmith R.
      • Robarge K.
      • Sutherlin D.
      • Scales S.J.
      • Gould S.E.
      • Yauch R.L.
      • de Sauvage F.J.
      Small molecule inhibition of GDC-0449 refractory smoothened mutants and downstream mechanisms of drug resistance.
      • Robinson G.W.
      • Orr B.A.
      • Wu G.
      • Gururangan S.
      • Lin T.
      • Qaddoumi I.
      • Packer R.J.
      • Goldman S.
      • Prados M.D.
      • Desjardins A.
      • Chintagumpala M.
      • Takebe N.
      • Kaste S.C.
      • Rusch M.
      • Allen S.J.
      • Onar-Thomas A.
      • Stewart C.F.
      • Fouladi M.
      • Boyett J.M.
      • Gilbertson R.J.
      • Curran T.
      • Ellison D.W.
      • Gajjar A.
      Vismodegib Exerts Targeted Efficacy Against Recurrent Sonic Hedgehog-Subgroup Medulloblastoma: Results From Phase II Pediatric Brain Tumor Consortium Studies PBTC-025B and PBTC-032.
      Thus, new targets to suppress oncogenic SHh ligand signaling are of interest. In a different context, activating the SHh pathway holds promise for developmental disorders where the native function of SHh is compromised by diminished expression. Mutations in the human SHh gene are implicated in holoprosencephaly (HPE), a devasting congenital disorder affecting fetal brain development.
      • Belloni E.
      • Muenke M.
      • Roessler E.
      • Traverso G.
      • Siegel-Bartelt J.
      • Frumkin A.
      • Mitchell H.F.
      • Donis-Keller H.
      • Helms C.
      • Hing A.V.
      • Heng H.H.
      • Koop B.
      • Martindale D.
      • Rommens J.M.
      • Tsui L.C.
      • Scherer S.W.
      Identification of Sonic hedgehog as a candidate gene responsible for holoprosencephaly.
      Roughly half of the reported HPE-associated mutations in SHh map to the gene’s 3’ region,
      • Hong S.
      • Hu P.
      • Jang J.H.
      • Carrington B.
      • Sood R.
      • Berger S.I.
      • Roessler E.
      • Muenke M.
      Functional analysis of Sonic Hedgehog variants associated with holoprosencephaly in humans using a CRISPR/Cas9 zebrafish model.
      encoding a partially characterized autoprocessing domain called SHhC. In this context, small molecules that enhance SHh signaling by restoring SHhC function could find therapeutic application.
      The autoprocessing SHhC domain is present in the precursor form of SHh where it self-catalyzes an essential biosynthetic step called cholesterolysis. Cholesterolysis occurs in the endoplasmic reticulum (ER) and results in the release and carboxyl-terminal cholesterylation of the adjacent SHh ligand domain (Scheme 1, left).
      • Bumcrot D.A.
      • Takada R.
      • McMahon A.P.
      Proteolytic processing yields two secreted forms of sonic hedgehog.
      • Porter J.A.
      • Young K.E.
      • Beachy P.A.
      Cholesterol modification of hedgehog signaling proteins in animal development.
      • Callahan B.P.
      • Wang C.
      Hedgehog Cholesterolysis: Specialized Gatekeeper to Oncogenic Signaling.
      The native reaction begins with a backbone peptide bond rearrangement at the junction between the SHh ligand and the SHhC domain. This rearrangement activates the terminal Gly residue of the SHh ligand as a thioester at the Cys (C1) side chain of SHhC. Mechanistic overlap exists here with self-splicing inteins.
      • Hall T.M.
      • Porter J.A.
      • Young K.E.
      • Koonin E.V.
      • Beachy P.A.
      • Leahy D.J.
      Crystal structure of a Hedgehog autoprocessing domain: homology between Hedgehog and self-splicing proteins.
      ,
      • Perler F.B.
      Protein splicing of inteins and hedgehog autoproteolysis: structure, function, and evolution.
      In a step unique to Hh family proteins, SHhC then catalyzes nucleophilic attack at the internal thioester by a cholesterol molecule. A conserved aspartate residue of SHhC, Asp46 (D46), is the putative general base for the attacking C3-OH group of cholesterol.
      • Xie J.
      • Owen T.
      • Xia K.
      • Callahan B.
      • Wang C.
      A Single Aspartate Coordinates Two Catalytic Steps in Hedgehog Autoprocessing.
      Cleavage releases cholesterylated SHh ligand for N-palmitoylation, Golgi transport, extracellular secretion and downstream cell/cell signaling. Mutations that deactivate SHhC result in SHh precursor retention in the ER and eventual removal through Endoplasmic Reticulum Associated Degradation (ERAD).
      • Chen X.
      • Tukachinsky H.
      • Huang C.H.
      • Jao C.
      • Chu Y.R.
      • Tang H.Y.
      • Mueller B.
      • Schulman S.
      • Rapoport T.A.
      • Salic A.
      Processing and turnover of the Hedgehog protein in the endoplasmic reticulum.
      • Huang C.H.
      • Hsiao H.T.
      • Chu Y.R.
      • Ye Y.
      • Chen X.
      Derlin2 protein facilitates HRD1-mediated retro-translocation of sonic hedgehog at the endoplasmic reticulum.
      • Tang H.Y.
      • Huang C.H.
      • Zhuang Y.H.
      • Christianson J.C.
      • Chen X.
      EDEM2 and OS-9 are required for ER-associated degradation of non-glycosylated sonic hedgehog.
      Engineered sterols have the potential to circumvent catalytic defects in SHhC and restore SHh ligand biosynthesis in some autoprocessing mutants (Scheme 1, right).(Ref
      • Ciulla D.A.
      • Jorgensen M.T.
      • Giner J.L.
      • Callahan B.P.
      Chemical Bypass of General Base Catalysis in Hedgehog Protein Cholesterolysis Using a Hyper-Nucleophilic Substrate.
      and this work)
      Figure thumbnail sc1
      Scheme 1Native Hedgehog SHhC autoprocessing with substrate cholesterol and non-native, induced autoprocessing using chemical rescue approach. Left. In the native cholesterolysis pathway, SHhC (green) activates then cleaves off the adjacent SHh ligand (blue) using cholesterol as the terminal nucleophile. Right Defective autoprocessing by SHhC point mutant, D46A (orange), is reactivated by engineered sterols, such as 2-α carboxy cholestanol (2-ACC), described here.
      Here we describe and validate the first homogenous live cell autoprocessing assay to screen for inhibitors and activators of human SHhC function. Chemical activators of SHhC could mitigate the impact of autoprocessing mutations, diminishing HPE pathology; antagonists of SHhC provide a novel means to block oncogenic biosynthesis of the Sonic Hh ligand.
      • Yauch R.L.
      • Dijkgraaf G.J.
      • Alicke B.
      • Januario T.
      • Ahn C.P.
      • Holcomb T.
      • Pujara K.
      • Stinson J.
      • Callahan C.A.
      • Tang T.
      • Bazan J.F.
      • Kan Z.
      • Seshagiri S.
      • Hann C.L.
      • Gould S.E.
      • Low J.A.
      • Rudin C.M.
      • de Sauvage F.J.
      Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma.
      ,
      • Amakye D.
      • Jagani Z.
      • Dorsch M.
      Unraveling the therapeutic potential of the Hedgehog pathway in cancer.
      Our method couples intracellular SHhC autoprocessing to the extracellular secretion of the ATP-independent nanoluciferase (NLuc) enzyme.
      • Hall M.P.
      • Unch J.
      • Binkowski B.F.
      • Valley M.P.
      • Butler B.L.
      • Wood M.G.
      • Otto P.
      • Zimmerman K.
      • Vidugiris G.
      • Machleidt T.
      • Robers M.B.
      • Benink H.A.
      • Eggers C.T.
      • Slater M.R.
      • Meisenheimer P.L.
      • Klaubert D.H.
      • Fan F.
      • Encell L.P.
      • Wood K.V.
      Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.
      A Sonic Hedgehog wild-type (WT) autoprocessing reporter line is established in HEK293 cells for evaluating SHhC inhibitors by a concentration-dependent loss of extracellular NLuc bioluminescence. We also describe and validate an inducible autoprocessing mutant (D46A) for identifying SHhC activators by the gain of extracellular NLuc bioluminescence. Direct NLuc detection in the media of 1536-well cell culture plates is facilitated by using methylphosphonic acid coelenterazine, CLZ-2P, a novel anionic substrate analog for NLuc.

      2. RESULTS:

      2.1 Reporter Strategy

      Autoprocessing activity of precursor Sonic Hh protein appears to reside exclusively in SHhC. The SHh ligand is a spectator to the transformation and this arrangement allows for the substitution of the SHh ligand with heterologous polypeptides. Chimeric, functional SHh reporter constructs have been described where green fluorescent protein is fused to SHhC, and more recently, where Halo-Tag is fused to SHhC.
      • Vincent S.
      • Thomas A.
      • Brasher B.
      • Benson J.D.
      Targeting of proteins to membranes through hedgehog auto-processing.
      ,
      • Mozhdehi D.
      • Luginbuhl K.M.
      • Dzuricky M.
      • Costa S.A.
      • Xiong S.
      • Huang F.C.
      • Lewis M.M.
      • Zelenetz S.R.
      • Colby C.D.
      • Chilkoti A.
      Genetically Encoded Cholesterol-Modified Polypeptides.
      We substituted the SHh ligand with HA-tagged nanoluciferase
      • Hall M.P.
      • Unch J.
      • Binkowski B.F.
      • Valley M.P.
      • Butler B.L.
      • Wood M.G.
      • Otto P.
      • Zimmerman K.
      • Vidugiris G.
      • Machleidt T.
      • Robers M.B.
      • Benink H.A.
      • Eggers C.T.
      • Slater M.R.
      • Meisenheimer P.L.
      • Klaubert D.H.
      • Fan F.
      • Encell L.P.
      • Wood K.V.
      Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.
      (HA-NLuc) with a view toward an HTS-compatible assay system.
      Three related HA-NLuc-SHhC fusion constructs were prepared (Figure 1 A). First, a wild-type (WT) HA-NLuc-SHhC was designed for assessment of native SHhC activity and to characterize potential autoprocessing inhibitors. Autoprocessing of the WT reporter construct is expected to cleave off and holesterylated HA-NLuc using cellular cholesterol in the ER. Cholesterylated HA-NLuc would then transit through the secretory pathway for extracellular release. NLuc activity present in the culture media thereby reports cellular SHhC autoprocessing. The N-terminal HA tag of NLuc enables the analysis of SHhC autoprocessing by Western blot. The WT reporter construct is intended for evaluating autoprocessing inhibitors by a decrease in extracellular NLuc and by an accumulation of intracellular WT precursor.
      Figure thumbnail gr1
      FIGURE 1Monitoring autoprocessing in live cells using HA-tagged nanoluciferase (HA-NLuc) as a secreted, proxy reporter of SHhC activity. A. Autoprocessing reporter constructs. WT, constitutively active autoprocessing using cellular cholesterol; C1A, irreversibly inactive autoprocessing mutant; D46A, conditional mutant with inducible autoprocessing activity. Reporter constructs carry an N-terminal HA epitope tag (grey) for Western analysis and the NLuc (blue) enzyme for non-invasive measurements of autoprocessing by media bioluminescence. B. Media bioluminescence reflects intracellular SHhC autoprocessing. Functional behavior of the WT, C1A, and D46A reporters was evaluated 18 hours after transient transfection in HEK293 cells (n=12). Inducibility of the D46A reporter was assessed by comparison of +/- the activator, 2-ACC (5 μM). No cholesterol was added above the cholesterol in the 10% FBS present the culture media. Statistical analysis: ANOVA followed by Tukey’s pairwise comparison using GraphPad Prism. C. Western blot for WT, C1A and D46A precursor and product using the N-terminal HA epitope. Cell extract from HEK293 cells was analyzed 18 h after transient transfection of the three reporter constructs in the absence (left) and presence (right) of 2-ACC (5 μM). GAPDH served as a loading control.
      We prepared a control construct, C1A, to mimic 100% inhibition of SHhC autoprocessing. This reporter fuses HA-NLuc to a C1A mutant of SHhC. The Cys>Ala mutation at position 1 of SHhC removes the critical nucleophilic thiol group required for thioester formation, thereby blocking SHhC enzymatic function. The precursor form of the C1A reporter is expected to remain largely in the cellular fraction where it would be subject to degradation by ERAD. Compounds that act specifically toward SHhC autoprocessing are not expected to influence bioluminescence from the catalytically inactive C1A or alter the reporter protein’s steady-state expression when analyzed by Western blot.
      In the third construct, we fused HA-NLuc to a D46A point mutant of SHhC for an autoprocessing reporter with inducible activity. The D46A construct is intended for evaluating small molecules as potential autoprocessing activators. Earlier studies on Drosophila Hh autoprocessing indicated that D46A mutants of HhC can form internal thioester; however, without the conserved active-site D46 carboxylate group, transesterification to substrate cholesterol is prevented.
      • Xie J.
      • Owen T.
      • Xia K.
      • Callahan B.
      • Wang C.
      A Single Aspartate Coordinates Two Catalytic Steps in Hedgehog Autoprocessing.
      The corresponding D46A mutation in human SHhC was expected to behave in a similar manner, blocking native cholesterolysis in cells. Based on our experiments with Drosophila HhC, we hypothesized that internal thioester generated by D46A could be resolved with rationally designed analogs such as 3-HPC (3-β hydroperoxycholestane) that can bypass the mutant’s catalytic defect.
      • Ciulla D.A.
      • Jorgensen M.T.
      • Giner J.L.
      • Callahan B.P.
      Chemical Bypass of General Base Catalysis in Hedgehog Protein Cholesterolysis Using a Hyper-Nucleophilic Substrate.
      For the in vitro cell culture work here, we prepared a more chemically stable hyper-nucleophilic analog, 2-α carboxy cholestanol (2-ACC). The carboxylate group vicinal to the attacking C3 hydroxyl group in 2-ACC was designed to functionally replace the missing carboxylate in the D46A mutant. (Scheme 1, right; and Figure 2). Below, we demonstrate that 2-ACC serves as an intracellular inducer of D46A.
      Figure thumbnail gr2
      FIGURE 2Substrate cholesterol for WT reporter autoprocessing and synthetic activator substrate, 2-ACC, for inducing autoprocessing by the D46A reporter.

      2.2 Nanoluciferase (NLuc) as a surrogate SHhN ligand and extracellular bioluminescent reporter

      Initial characterization of the SHhC autoprocessing reporters was carried out by transient transfection using HEK293 cells. HEK293 have been used before for cellular studies of human SHhC autoprocessing.
      • Traiffort E.
      • Dubourg C.
      • Faure H.
      • Rognan D.
      • Odent S.
      • Durou M.R.
      • David V.
      • Ruat M.
      Functional characterization of sonic hedgehog mutations associated with holoprosencephaly.
      ,
      • Cooper M.K.
      • Porter J.A.
      • Young K.E.
      • Beachy P.A.
      Teratogen-mediated inhibition of target tissue response to Shh signaling.
      The WT, C1A and D46A chimeric precursors were cloned into the pDisplay (Invitrogen) mammalian expression vector in frame with the vector’s N-terminal Ig κ-chain secretion sequence and HA epitope tag. Following transfection in HEK293, SHhC activity was assayed by NLuc bioluminescence in the culture media and by Western blot of the cell extract.
      Figure 1B summarizes media bioluminescence using the NLuc NanoGlo reagent (Promega) following transient transfections with the three reporter plasmids. Samples were collected and analyzed 18 hours after transfection. The first column in Figure 1B (green circles) shows readings from the WT samples. The mean bioluminescence value for the WT transfected cells was consistently the highest of the four conditions tested here, in accord with fully active SHhC autoprocessing. The catalytic mutant, C1A (Figure 1B, red squares) by contrast had the lowest output, reduced from WT by 6.5-fold, consistent with defective autoprocessing and intracellular precursor retention.
      • Chen X.
      • Tukachinsky H.
      • Huang C.H.
      • Jao C.
      • Chu Y.R.
      • Tang H.Y.
      • Mueller B.
      • Schulman S.
      • Rapoport T.A.
      • Salic A.
      Processing and turnover of the Hedgehog protein in the endoplasmic reticulum.
      The last two columns in Figure 1B show the media bioluminescence from HEK293 cells transfected with the D46A reporter plasmid (–) and (+) 2-ACC (5 μM). In samples lacking 2-ACC, the D46A samples produced media bioluminescence that is on par with the C1A reporter. This correspondence is consistent with the inability of D46A to autoprocess using cellular cholesterol. The last column in Figure 1B showing D46A (+) 2-ACC indicates a clear gain of media bioluminescence (4.6-fold) relative to D46A without 2-ACC. The enhanced signal is exciting and supports the induction of autoprocessing in HEK293 cells by added 2-ACC. Examples like this where “chemical rescue” of a mutant enzyme that occurs in the cell are rare.
      • Qiao Y.
      • Molina H.
      • Pandey A.
      • Zhang J.
      • Cole P.A.
      Chemical rescue of a mutant enzyme in living cells.
      • Lowry W.E.
      • Huang J.
      • Ma Y.C.
      • Ali S.
      • Wang D.
      • Williams D.M.
      • Okada M.
      • Cole P.A.
      • Huang X.Y.
      Csk, a critical link of g protein signals to actin cytoskeletal reorganization.
      • Marletta M.A.
      Raising enzymes from the dead and the secrets they can tell.
      Induced cellular autoprocessing of D46A by 2-ACC is further supported by Western blot analysis, described next. In the SI material, we also report that the corresponding D46A mutant of Drosophila HhC has its defective autoprocessing activity restored by 2-ACC (Supplementary Figure 1). Bioluminescence was not appreciably enhanced when 2-ACC was added to cells transfected with the C1A or WT reporters, consistent with the specificity of this induction. Overall, the results of the transient transfection experiments in Figure 1B provide proof-of-concept for the WT, C1A and D46A reporters.
      To complement the comparison based on NLuc bioluminescence, we probed the corresponding cellular fractions of WT, C1A and D46A by Western blot using the construct’s HA epitope tag (Figure 1C). Lysate samples were denatured, separated by SDS-PAGE and probed with a monoclonal HA-HRP antibody (Sigma) to identify precursor protein, HA-NLuc-SHhC (52 kDa) and autoprocessing product, HA-NLuc (24.4 kDa). Analysis of samples with the WT reporter confirmed SHhC autoprocessing activity, with precursor and product in a ratio of ∼1 to 10. Lysate from the C1A samples showed signal for precursor only, consistent with deactivated SHhC autoprocessing. In D46A, induction of autoprocessing was evident by comparison of samples (-)/(+) 2-ACC. In the absence of 2-ACC, the D46A precursor is expressed without appreciable generation of autoprocessing product. By contrast, signal for the D46A precursor decreases in the (+) 2-ACC sample and the HA-NLuc product signal accumulates, consistent with restored SHhC autoprocessing. The D46A induction was shown to be specific to 2-ACC (Figure 1C, compare -/+ 2-ACC sample sets for D46A with WT and C1A, and vide infra).

      2.3 Miniaturizing the NLuc-SHhC autoprocessing reporter system for 1536 well plates

      With a view toward applications to high throughput chemical screening for inhibitors and activators
      • Inglese J.
      • Johnson R.L.
      • Simeonov A.
      • Xia M.
      • Zheng W.
      • Austin C.P.
      • Auld D.S.
      High-throughput screening assays for the identification of chemical probes.
      of SHhC we sought to streamline assay setup by first preparing stable WT and D46A reporter cell lines. CRISPR/Cas9 targeted gene insertion
      • He X.
      • Tan C.
      • Wang F.
      • Wang Y.
      • Zhou R.
      • Cui D.
      • You W.
      • Zhao H.
      • Ren J.
      • Feng B.
      Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.
      was used to integrate the two reporter constructs into HEK293 cells (Materials and Methods).
      In line with the transient transfection results above, the stable WT line showed constitutive autoprocessing with strong bioluminescence output in the culture media. We found that treatment of the WT line with brefeldin A, a protein secretion blocker that disrupts ER to Golgi transport, attenuated media bioluminescence, supporting the notion that Nluc activity in the media largely reflects secretory pathway transport rather than non-specific cell leakage (Fig. 3A). Bioluminescence in the lysate fraction remained relatively constant, indicating sustained expression of the WT construct in brefeldin A treated cells (Fig. 3A, inset). For additional validation, we tested whether the WT reporter would recapitulate the ERAD/proteasome sensitivity observed with native human Sonic Hh precursor.
      • Chen X.
      • Tukachinsky H.
      • Huang C.H.
      • Jao C.
      • Chu Y.R.
      • Tang H.Y.
      • Mueller B.
      • Schulman S.
      • Rapoport T.A.
      • Salic A.
      Processing and turnover of the Hedgehog protein in the endoplasmic reticulum.
      Studies by Salic et al suggest that the human SHh precursor in the ER is subjected to a dynamic partitioning between native autoprocessing and ERAD-mediated proteasome degradation. We monitored WT precursor levels by Western blot following various treatments. We first treated the WT line with the protein synthesis inhibitor cycloheximide (CHX). With CHX, we observed a time dependent precursor loss (Fig. 3B, CHX, top row of Western blot), which could be attributed to native autoprocessing as well as to ERAD. For comparison, in the untreated (DMSO only) samples, the HA-NLuc-SHhC precursor appears to be at a steady state (Fig. 3B, no treatment). To separate the influence of native autoprocessing from ERAD degradation, the WT line was treated initially with the proteasome inhibitor MG132. Inhibiting the proteasome resulted in a ∼70% increase in the precursor signal. Precursor accumulation is consistent with diminished ERAD (Fig. 3B, MG132). Next, the WT cells were treated with a combination of CHX and MG132. Here we observed a more gradual consumption of precursor (Fig. 3B, MG132+CHX). Under this final condition, precursor levels should predominately reflect SHhC autoprocessing with endogenous cholesterol.
      • Chen X.
      • Tukachinsky H.
      • Huang C.H.
      • Jao C.
      • Chu Y.R.
      • Tang H.Y.
      • Mueller B.
      • Schulman S.
      • Rapoport T.A.
      • Salic A.
      Processing and turnover of the Hedgehog protein in the endoplasmic reticulum.
      Overall, the pattern of accumulation and depletion of the WT precursor protein in Figure 3B agrees with the dynamic partitioning between ERAD-mediated destruction and SHhC autoprocessing observed with native SHh precursor.
      Figure thumbnail gr3
      FIGURE 3Validation of the WT line as a reporter for native SHhC autoprocessing. A. Extracellular secretion of NLuc by WT reporter blocked by Brefeldin A. Media bioluminescence was measured 1, 2, 3, and 6 hours after addition of the indicated concentrations of Brefeldin A. (inset) Bioluminescence from samples of the corresponding cell lysate fraction after 6 hr Brefeldin A treatment. B. Intracellular WT reporter protein response to cycloheximide (CHX) and MG132. Upper. Western Blot analysis. Control samples treated with DMSO control; 25 μg/ml cycloheximide; 50 μM MG132; and co-treated with 25 μg/ml CHX and 50 μM MG132. Lower. Plot of WT precursor abundance using Western blot images shown based on analysis with Image J.
      The stable HEK293 D46A reporter line displayed specific, 2-ACC inducible autoprocessing activity as measured by extracellular NLuc activity and in accord with the transient transfection experiments. In the absence of 2-ACC, the D46A reporter line again displayed low media bioluminescence, consistent with intracellular retention. As shown in Figure 4A, there was a gain of media bioluminescence with added 2-ACC that was concentration-dependent and saturable over an 11-pt titration, from 20 nM to 20 μM in 2-ACC. Analysis of the concentration-response data by non-linear regression yielded an EC50 value of 1.6 μM for 2-ACC. As an additional control, we added cholesterol to the D46A line at a concentration equivalent to 2-ACC (5 μM); no increase in media bioluminescence was apparent (Figure 4B). The hyper-nucleophilic 3-HPC failed to produce measurable induction of cellular autoprocessing by D46A, likely a consequence of the hydroperoxide groups instability.
      Figure thumbnail gr4
      FIGURE 4Validation of the D46A line as a chemically inducible SHhC autoprocessing reporter. A. Concentration-response activation curve for 2-ACC based on media NLuc activity from D46A (n=4). EC50 = 1.6 μM. B. Induced autoprocessing with D46A is 2-ACC specific. Endogenous cholesterol supplied by the 10% FBS – no additional cholesterol added (no treatment); additional cholesterol added (5 μM, final), added 3-HPC (5 μM, final) compared with added 2-ACC (5 μM, final) as potential inducers of D46A autoprocessing (n=21). Statistical analysis: ANOVA, Tukey’s pairwise comparison (GraphPad Prism).
      Next, we focused on optimizing and miniaturizing the WT/D46A reporter system to the 1536-well format. To streamline the assay, we designed and synthesized the anionic coelenterazine analog (CLZ-2P) (Fig. 5A). Traditional imidazopyrazinone nanoluciferase substrates such as coelenterazine (CLZ-H) and furimazine (NanoGlo) are uncharged and membrane permeable and therefore require careful media transfer from the cell culture plate to an assay plate to separate signal from intra- and extracellular NLuc.
      • Iannotti M.J.
      • MacArthur R.
      • Jones R.
      • Tao D.
      • Singec I.
      • Michael S.
      • Inglese J.
      Detecting Secretory Proteins by Acoustic Droplet Ejection in Multiplexed High-Throughput Applications.
      Intracellular NLuc could include product and precursor forms of the SHhC reporter proteins used here, confounding analysis of autoprocessing activity; whereas extracellular NLuc is expected to reflect autoprocessing product only. CLZ-2P, incorporating the anionic methylphosphonic acid at the 2-(4-hydroxybenzyl) moiety of CLZ-H was devised to eliminate cell permeability and the media transfer step for measurement of media bioluminescence. The C-P phosphonyl bond in CLZ-2P stabilizes the anionic group against P-O cleaving phosphatases, similar to the previously described analog described by Lindberg et al
      • Lindberg E.
      • Mizukami S.
      • Ibata K.
      • Fukano T.
      • Miyawakide A.
      • Kikuchi K.
      Development of cell-impermeable coelenterazine derivatives.
      , but synthetically more tractable for quantities needed in HTS applications. The data in Figure 5B, 6 and Supplementary Figure 4 supports CLZ-2P as an alternative NLuc substrate that is suited for mix-and-read assays of the WT and D46A in 1536 well plates.
      Figure thumbnail gr5
      FIGURE 5Validation of CLZ-2P for mix-and-read measurements of extracellular NLuc activity. A. Anionic coelenterazine, CLZ-2P. B. Samples of WT and D46A with added CLZ-2P (top) or coelenterazine (CLZ-H) (bottom) were analyzed as a function of increasing 2-ACC in 1536-well plates. Data were row-wise normalized to DMSO neutral control at each time point. Error bars represent standard deviation of the average of 2 replicate wells.
      Figure thumbnail gr6
      FIGURE 6Concentration-response curves for WT and D46A in 1536-well plates with control compounds. Graphs for 2-ACC (left) and cycloheximide (middle) titrations are based on NLuc bioluminescence in the media using CLZ-2P as NLuc substrate. Digitonin experiments (right) are based on cytotoxicity, measured by the CellTiter-Glo reagent. Curves were fit in GraphPad Prism and error bars represent the standard deviation of 2 replicate wells for each concentration across the respective assay plates. n=2.
      Analysis of the WT and D46A reporter lines in the 1536-well plate format was conducted hereafter using CLZ-2P. We observed a relatively low coefficient of variation of 4-5% between 32 intra-plate replicates for both cell lines (Supplementary Table 1), demonstrating well-to-well reproducibility in this assay format. Both reporter lines yielded a suitable Z’ factor, with WT = 0.83 and D46A = 0.64, using cycloheximide and 2-ACC, respectively.
      • Inglese J.
      • Johnson R.L.
      • Simeonov A.
      • Xia M.
      • Zheng W.
      • Austin C.P.
      • Auld D.S.
      High-throughput screening assays for the identification of chemical probes.
      In Figure 6 A-B, the concentration-response curves are shown for D46A and WT treated with 2-ACC and CHX. In agreement with the experiments above, activation by 2-ACC is specific to D46A, with an EC50 of ∼5 μM and ∼100% activation at 28.75 μM 2-ACC. Extracellular secretion of NLuc by WT was also shown to be sensitive to CHX treatment, with maximum inhibition of NLuc activity in the media at 57.5 μM CHX. As an additional test, cells were treated with the membrane-disrupting digitonin toxin and cytotoxicity was measured with CellTiter-Glo assay reagent. D46A and WT responded near identically, with maximum lethal digitonin concentration of 115 μM, Z’ factor:0.87 (Fig. 6C). Taken together, the robust performance of the two stable lines in HTS-compatible plates, along with the efficacy of CLZ-2P as an NLuc substrate for media bioluminescence measurements, encourage the application of this novel reporter system to identify the first chemical probes of SHhC autoprocessing.

      DISCUSSION:

      Early biochemical studies on chicken SHh protein demonstrated “proteolytic processing” into two SHh products of approximately 19 kDa (SHh ligand) and 27 kDa (SHhC).
      • Bumcrot D.A.
      • Takada R.
      • McMahon A.P.
      Proteolytic processing yields two secreted forms of sonic hedgehog.
      Work carried out around the same time with mouse SHh reported similar behavior.
      • Chang D.T.
      • Lopez A.
      • von Kessler D.P.
      • Chiang C.
      • Simandl B.K.
      • Zhao R.
      • Seldin M.F.
      • Fallon J.F.
      • Beachy P.A.
      Products, genetic linkage and limb patterning activity of a murine hedgehog gene.
      Both studies involved transient expression of the full-length SHh precursor in cultured eukaryotic cells followed by Western blotting of cell lysate. Gel-based assays remain the standard approach to monitor cellular SHh autoprocessing despite the method’s idiosyncrasies, expense and low throughput. Cell-free and gel-free autoprocessing assays using recombinant protein, which we recently devised for kinetic studies of Drosophila Hh autoprocessing,
      • Jiang S.Q.
      • Paulus H.
      A high-throughput, homogeneous, fluorescence polarization assay for inhibitors of hedgehog protein autoprocessing.
      ,
      • Owen T.S.
      • Ngoje G.
      • Lageman T.J.
      • Bordeau B.M.
      • Belfort M.
      • Callahan B.P.
      Forster resonance energy transfer-based cholesterolysis assay identifies a novel hedgehog inhibitor.
      have so far resisted application to the human Hh homologs. In our hands, only sparing amounts of bacterially expressed human SHhC can be recovered, and that material seems to lack autoprocessing activity. Requirements for N-linked glycosylation
      • Huang C.H.
      • Hsiao H.T.
      • Chu Y.R.
      • Ye Y.
      • Chen X.
      Derlin2 protein facilitates HRD1-mediated retro-translocation of sonic hedgehog at the endoplasmic reticulum.
      and reductive activation
      • Chen X.
      • Tukachinsky H.
      • Huang C.H.
      • Jao C.
      • Chu Y.R.
      • Tang H.Y.
      • Mueller B.
      • Schulman S.
      • Rapoport T.A.
      • Salic A.
      Processing and turnover of the Hedgehog protein in the endoplasmic reticulum.
      of SHhC in the mammalian ER offer a possible explanation.
      The in vitro live cell SHhC activity assay reported here represents an important resource for future studies of human Hh protein autoprocessing. With NLuc as the surrogate SHh ligand, intracellular SHhC autoprocessing is coupled to the secretion of a thermostable, ATP-independent bioluminescent reporter. Measurement of NLuc enzymatic activity is carried out non-invasively in a semi-quantitative manner in culture media. We show that bioluminescent output is sufficiently robust for miniaturization to 1536 well plates. Inhibitors of SHhC autoprocessing are expected to suppress NLuc secretion from cells expressing the WT reporter, while activators of SHhC autoprocessing are identified by enhanced NLuc secretion from the D46A reporter. The development of CLZ-2P as an impermeable substrate for NLuc increases assay throughput. With CLZ-2P, measurements of secreted NLuc are completed as a simple mix-and-read step in the presence of live cells.
      A modified version of the FRET-based biochemical reporter of HhC autoprocessing (Ref 42) was considered initially for the purpose of monitoring intracellular human SHhC autoprocessing. We elected to pursue bioluminescence from NLuc instead of using a FRET reporter for several reasons: the greater sensitivity possible with the enzyme amplified output signal from NLuc; the smaller size of NLuc (19 kDa) compared to a FRET-active pair of fluorescent proteins (60 kDa); and the expectation of a more straightforward means of signal measurement and greater dynamic range of that signal with the NLuc reporter compared with a ratiometric FRET reporter.
      The WT and D46A reporter cell lines are poised for quantitative high-throughput screening (qHTS)
      • Inglese J.
      • Auld D.S.
      • Jadhav A.
      • Johnson R.L.
      • Simeonov A.
      • Yasgar A.
      • Zheng W.
      • Austin C.P.
      Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries.
      to support the search for chemical probes of this key initiating step in the Sonic Hh signaling pathway. Autoprocessing events like Hh protein cholesterolysis that are single turnover and partly unimolecular represent challenging but not untenable targets for small molecules.
      • Owen T.S.
      • Ngoje G.
      • Lageman T.J.
      • Bordeau B.M.
      • Belfort M.
      • Callahan B.P.
      Forster resonance energy transfer-based cholesterolysis assay identifies a novel hedgehog inhibitor.
      ,
      • Wagner A.G.
      • Stagnitta R.T.
      • Xu Z.
      • Pezzullo J.L.
      • Kandel N.
      • Giner J.L.
      • Covey D.F.
      • Wang C.
      • Callahan B.P.
      Nanomolar, Noncovalent Antagonism of Hedgehog Cholesterolysis: Exception to the "Irreversibility Rule" for Protein Autoprocessing Inhibition.
      • Smith C.J.
      • Wagner A.G.
      • Stagnitta R.T.
      • Xu Z.
      • Pezzullo J.L.
      • Giner J.L.
      • Xie J.
      • Covey D.F.
      • Wang C.
      • Callahan B.P.
      Subverting Hedgehog Protein Autoprocessing by Chemical Induction of Paracatalysis.
      • Xie J.
      • Owen T.
      • Xia K.
      • Singh A.V.
      • Tou E.
      • Li L.
      • Arduini B.
      • Li H.
      • Wan L.Q.
      • Callahan B.
      • Wang C.
      Zinc inhibits Hedgehog autoprocessing: linking zinc deficiency with Hedgehog activation.
      • Owen T.S.
      • Xie X.J.
      • Laraway B.
      • Ngoje G.
      • Wang C.
      • Callahan B.P.
      Active site targeting of hedgehog precursor protein with phenylarsine oxide.
      Antagonists of SHhC autoprocessing are sought to inhibit Sonic Hh biosynthesis, blocking the ligand from oncogenic signaling in sporadic tumors. In developmental disorders like HPE where SHh ligand biosynthesis is compromised by congenital mutation, activators of SHhC could serve as chemical chaperones to restore SHh signaling.

      Experimental Procedures

      Biochemicals and cell biology reagents

      DNA restriction enzymes, T4 DNA ligase and chemically competent DH5 alpha cells (NEB); Alt-R CRISPR-Cas9 crRNA, Cas9 expression plasmid, and PCR oligos (IDT). HA-HRP primary antibody; GAPDH-HRP primary antibody (Santa Cruz SC-365062); Hyclone 0.25% Trypsin-EDTA (GE Life Sciences); penicillin/streptomycin, EMEM, FBS (Corning); G418, OptiMEM I (Gibco); digitonin, MG132, cycloheximide (Millipore Sigma); HEK293 cells (ATCC, provided by Tracy Brooks, SUNY Binghamton). Plates: 96 Well Black Polystyrene Microplate (Corning 3650), 12 Well Nunclon Delta Surface (Thermo Scientific), 96 Well Tissue Culture Plate (VWR 734-2327), 50 mL Tissue Culture Flask Blue Vented Cap (Corning 353108), white solid bottom TC treated 1536 well plate (Greiner Bio-One 789173-F).

      Reporter Constructs

      Chimeric SHh precursor encoding HA-tagged NLuc fused to native and mutant forms of human Sonic HhC autoprocessing domain were assembled in pDisplay (Invitrogen). Fragments encoding Sonic HhC were inserted into pDisplay using PstI and XhoI; codon optimized NLuc was inserted using the vector’s BglII and PstI sites. An oligo cassette for CRISPR-Cas9 genomic integration was introduced into the vector at a unique SnaBI site. Plasmids were purified for transfection with the ZymoPURE II plasmid midiprep kit (Zymoresearch). We thank Dr. Erich Roessler (NIH) for providing a Sonic Hh cDNA plasmid. Supplementary Figure 2 shows the amino acid sequence of assembled WT chimeric construct.

      Transient Transfection

      HEK293 cells were seeded into a 12-well plate at 100,000 cell/mL in complete EMEM supplemented with 10% FBS and 0.1 % Pen/Strep. When cells reached 70% confluency, media was replaced with reduced serum OptiMEM I containing lipofectamine-2000 with 1.6 μg/well reporter plasmid. After a one-hour transfection, media was replaced with complete EMEM and the cells were incubated overnight. Cells and media were then collected for bioluminescence assays and Western blot analysis.

      HEK293 Stable Cell Line Generation using CRISPR/CAS9

      We followed the general method outlined by He et al. to generate WT and D46A reporter lines.
      • He X.
      • Tan C.
      • Wang F.
      • Wang Y.
      • Zhou R.
      • Cui D.
      • You W.
      • Zhao H.
      • Ren J.
      • Feng B.
      Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.
      Nucleic acid fragments used for CRISPR/CAS9 integration are found in Supplementary Figure 3. HEK293 cells were cultured at 37°C 5% CO2 in EMEM supplemented with 10% FBS and 0.1% Pen/Strep. Cells were grown to 70% confluency in a 12-well plate. The media was replaced with reduced serum OptiMEM I containing lipofectamine-2000 (Invitrogen), the pDisplay reporter plasmid (0.6 μg), Cas9 expression plasmid (0.6 μg), and short guide RNA (0.4 μg). After one hour, the transfection media was replaced with complete media. Following an overnight recovery in complete EMEM, the media was exchanged with complete EMEM containing G418 antibiotic (400 μg/ml). Media was then replaced every 2-3 days and cells were passaged weekly. Bioluminescence measurements were made periodically to confirm expression of the reporter constructs. After four weeks under G418 selection, cells were diluted into 96-well plates at single cell per well density and then expanded into stable cell lines.

      Cell lysis and Western blot analysis

      HEK293 cells transfected with WT, C1A or D46A reporter plasmids were lysed after media aspiration by 250 μl ice-cold RIPA buffer (Thermo Scientific). After agitation by orbital shaking for 20 minutes at room temperature, the lysate suspension was collected into microcentrifuge tubes, treated with DNAse I (12 ng/μl) on ice for ten minutes, followed by centrifugation at 18,000 g for 5 minutes at 4°C. The supernatant was transferred to a clean tube and used immediately or stored at -80 °C. For Western analysis, aliquots of the soluble lysate were denatured by boiling at 95°C for 5 minutes in SDS-PAGE load buffer (final conc. Bromophenol blue 0.1%, glycerol 10%, SDS 2%, DTT 100 mM, Tris-HCl 50 mM). Denatured proteins were resolved by 12% SDS-PAGE. Following electrophoresis, the gel was submerged for 20 minutes in transfer buffer (Tris 2.5 mM, glycine 19 mM, 20% methanol, pH 8.3) then electroblotted (20 V) overnight at 4°C onto a 0.2 μm PVDF membrane (Amersham Hybond). Following transfer, the membrane was blocked with 5% BLOT-Quick at room temperature with gentle shaking for one hour. After rinses with TBST buffer (Tris 2 mM, NaCl 15 mM, Tween-20 0.1%), a solution of TBST/5% BLOT-quick with αHA-HRP (1/500) or αGAPDH-HRP (1/1000) was added. After gentle shaking at 4 °C, overnight, the membrane was rinsed 3x with TBST buffer, followed by a 15-minute wash, and then three five-minute washes with TBST buffer. Visualization of HRP was accomplished by colorimetric detection (Femto-Chromo HRP kit, G-Biosciences).

      Extracellular NLuc Assay for induced autoprocessing in the D46A line

      Stable D46A cells were seeded into 96-well plates at a density of 2x10
      • Bissey P.A.
      • Mathot P.
      • Guix C.
      • Jasmin M.
      • Goddard I.
      • Costechareyre C.
      • Gadot N.
      • Delcros J.G.
      • Mali S.M.
      • Fasan R.
      • Arrigo A.P.
      • Dante R.
      • Ichim G.
      • Mehlen P.
      • Fombonne J.
      Blocking SHH/Patched Interaction Triggers Tumor Growth Inhibition through Patched-Induced Apoptosis.
      cells/well (total volume 100 μl) and grown to ∼70% confluency over 72 hours. Seeding media contained 2-ACC, cholesterol, or 3-HPC, titrated over an 11-point 1:2 dilution from 20 μM to 20 nM. At least four intra-plate replicates were included for each concentration of sterol. We assayed 10 μl of the media for bioluminescence using the NanoGlo detection reagent (furimazine). We first prepared a NLuc reaction stock solution by combining 980 μl NanoGlo buffer with 20 μl of furimazine substrate. This solution was incubated in the dark at room temperature for five minutes. During this period, we prepared the bioluminescence assay plate. Each sample well in the assay plate (Corning 3650) contained 90 μl of distilled water and 10 μl media from the cell culture plate. To initiate bioluminescence, 10 μl of the NanoGlo reaction solution was added to each sample well in the assay plate, incubated with gentle shaking for two minutes, then assayed for bioluminescence using a Biotek Synergy H1 plate reader with gain equal to 150.

      Assay miniaturization and NLuc substrate optimization

      HEK293 cells expressing wildtype HA-NLuc-SHhC (WT) and HA-NLuc-SHhC (D46A) were grown in Eagle’s Minimum Essential Medium (ATCC) supplemented with 10% FBS (HyClone), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) in the absence of selection antibiotics. Cells were trypsinized from culture flasks, counted, diluted in growth media and plated at 1000, 1500, or 2000 cells per 4 μl per well in respective columns of white, solid bottom TC treated 1536-well plates (Greiner Bio-One) with a multidrop combi dispenser (ThermoFisher). Control compound digitonin was prepared at 20 mM stock concentration in DMSO and diluted in a 16-pt, 1:2 dilution, while MG132 and cycloheximide were prepared at a stock concentration of 10 mM in DMSO and titrated in 16-pt, 1:3 dilution. Each compound titration was transferred in duplicate to respective columns of assay plates along with replicate columns of DMSO vehicle control to seeded cells at 25 nL per well with a Mosquito liquid handler (SPTLabTech) immediately after cells were plated. Cells were covered with weighted metal lids with gas exchange pores (Wako) and incubated at 37°C, 5% CO2 and 95% humidity. After 24 hr 1 μl media was manually transferred from each well of one assay plate to a recipient white, solid bottom TC treated 1536-well plate with 3 μl 1x PBS predispensed in each well. NanoGlo luciferase assay substrate was prepared at a 1:100 dilution according to manufacturer’s protocol and 3 μl NanoGlo substrate was added to each well of media transfer plate and cell source plate with a BioRAPTR FRD (Beckman Coulter). Nanoluciferase luminescence for each plate was read on a ViewLux plate reader (PerkinElmer) after 5 min. Coelenterazine-H substrate was prepared as a 2 mM stock solution in 100% ethanol and coelenterazine-2P was diluted to a 2 mM stock solution in DMSO. Both substrates were prepared in a non-lytic PBS assay buffer (300 mM sodium ascorbate, 5 mM NaCl) at a working dilution of 20 μM. Coelenterazine substrates were added at 4 μl/well to one cellular assay plate each with a BioRAPTR FRD and nanoluciferase luminescence was read on a ViewLux plate reader within 2-5 min.

      qHTS 2-ACC Time Course Concentration-response Validation

      Stable HEK293 WT and D46A cells were grown and plated at 1.5x10
      • Liu Z.
      • Xu J.
      • He J.
      • Zheng Y.
      • Li H.
      • Lu Y.
      • Qian J.
      • Lin P.
      • Weber D.M.
      • Yang J.
      • Yi Q.
      A critical role of autocrine sonic hedgehog signaling in human CD138+ myeloma cell survival and drug resistance.
      cells/well (4 μl) in respective columns of six replicate white, solid bottom TC treated 1536-well plates as before. 2-α carboxy cholestanol was diluted in a 16-pt, 1:2 dilution starting at a high concentration of 10 mM and was added to the plated cells as above. Cells were incubated at 37°C, 5% CO2 and 95% humidity for 24-72 hr. Using the Mosquito liquid handler, 300 nl of media was transferred from each well of two assay plates to respective white, solid bottom TC treated 1536-well recipient plates containing 4 μl/well 1x PBS every 24 hr. One media transfer plate received 3 μl/well NanoGlo substrate while the corresponding cell source plate received 4 μl/well coelenterazine-2P substrate. Luminescence was read on a ViewLux plate reader after a 5 min incubation. Coelenterazine-H was added at 4 μl/well to the second set of plates and luminescence was read after 5 min.

      qHTS Data Analysis

      Assay statistics were calculated for 32 replicate wells of DMSO vehicle control and 2 wells of high concentration of respective compound treatment for initial assay optimization across the cell lines, cell densities, and for each substrate across a 72 hr time course. Statistics were then calculated for 64 wells of DMSO vehicle control and 32 replicate wells of max concentration of the respective control compounds for optimal assay conditions of 1500 cells/well treated for 48 hr. . Concentration response curves were determined for each compound and data was normalized to DMSO neutral control for each cell density for each cell line. Concentration response curves were fit and EC50 values calculated in Prism 7.0 (GraphPad, La Jolla, CA) with nonlinear regression log(agonist) vs. response – variable slope (four parameters) fit. Bell-shaped curves were also fit in Prism 7.0 with nonlinear regression and the equation:
      Y=S0+(S1S0)(1+10((logEC50X)×HillSlope1))+(S2S1)(1+10((logEC50X)×HillSlope2))


      2-ACC Synthesis

      Figure thumbnail fx1

      3β-Hydroxycholestane-2α-carboxylic acid (III)

      To a solution of IIa/b (22.0 mg mixed isomers, 0.048 mmol) in DCM (0.5 mL) was added 10% NaOH/MeOH (2.5 mL) and the solution was heated under reflux for 16 h. After cooling to rt, 10% hydrochloric acid (5 mL) was added, the mixture was extracted thoroughly with EtOAc, and the extracts were filtered through Na2SO4. Evaporation under reduced pressure yielded the product (20.6 mg, 100%). The diastereomers were separated by preparative TLC (250 μm silica plate, hexane / EtOAc 1:2 + 5% AcOH), and eluted with 2% AcOH/EtOAc. The more polar band contained the desired product. The procedure was repeated with compound IIc to yield an identical sample of III. 1H NMR (800 MHz, CDCl3): 3.818 (1H, td, J = 4.7, 10.6 Hz), 2.513 (1H, td, J = 3.0, 11.9 Hz), 2.048 (1H, dd, J = 3.2, 13.8 Hz), 1.965 (1H, dt, J = 3.0, 12.7 Hz), 1.809 (1H, m), 1.658 (2H, m), 1.58-1.47 (3H, m), 1.44-0.94 (m), 0.896 (3H, d, J = 6.4 Hz), 0.862 (6H, dd, J = 3.7, 6.6 Hz), 0.855 (3H, s), 0.673 (1H, td, J = 3.6, 11.4 Hz), 0.650 (3H, s). 13C NMR (201 MHz): 179.57, 71.14, 56.40, 56.23, 54.03, 46.79, 44.50, 42.55, 40.05, 39.87, 39.51, 36.16, 36.07, 35.78, 35.44, 31.84, 28.26, 28.21, 28.01, 24.18, 23.82, 22.81, 22.55, 21.30, 18.67, 12.52, 12.07. HRMS (HESI) m/z [M – H]-: Calcd for C28H47O3 431.3525; Found 431.3532. (NMR spectra for I, II, III, see Supplementary Figure 5)

      CLZ-2-methylphosphonic acid (CLZ-P) synthesis

      General Methods

      All air or moisture-sensitive reactions were performed under a positive pressure of argon with oven-dried glassware. 4M HCl in dioxane and cesium carbonate were purchased from Sigma-Aldrich and used as such. Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Method: A 7-minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8-minute run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3-micron, 3 x 75 mm) was used at a temperature of 50 °C. A Phenomenex Gemini Phenyl column (3-micron, 3 x 100 mm) was used at a temperature of 50 °C. Purity determination was performed using an Agilent Diode Array Detector for both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. 1H NMR spectra were recorded on Varian 400 MHz spectrometers. Chemical shifts are reported in ppm with undeuterated solvent (DMSO-d6 at 2.49 ppm) as an internal standard for DMSO-d6 solutions. High-resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).

      Synthesis of diethyl ((4-(3,3-diethoxy-2-oxopropyl)phenoxy)methyl)phosphonate (3)

      Figure thumbnail fx4
      An oven-dried flask containing 1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (0.794 g, 3.33 mmol, 1 eq) in acetonitrile (10 mL) was added cesium carbonate (1.628 g, 5.00 mmol, 1.5 eq). After stirring for 10 minutes at room temperature, (diethoxyphosphonyl)methyl trifluoromethanesulfonate (1 g, 3.33 mmol, 1 eq) was added and stirred at room temperature overnight. The reaction mixture was filtered through a pad of celite and washed with ethyl acetate. The filtrate was concentrated, and the residue was directly loaded to a flash 40 g silica column. The crude product was purified on a flash system eluting with 0-100 % ethyl acetate in hexanes over 32 column volumes to yield 0.62 g (48 %) pure product 3 after evaporating and drying process under high vacuum. LCMS Retention time = 5.3 min. (M+H)+ for C18H30O7P = 389.2.

      Synthesis of CLZ-2-methylphosphonic acid (CLZ-2P), aka NCATS-SM1462

      Figure thumbnail fx5
      An oven-dried round-bottomed flask containing a mixture of diethyl ((4-(3,3-diethoxy-2-oxopropyl)phenoxy)methyl)phosphonate 3 (0.617 g, 1.589 mmol, 1.2 eq) and 3-benzyl-5-(4-(2-(trimethylsilyl)ethoxy)phenyl)pyrazin-2-amine 4 (0.5 g, 1.324 mmol, 1 eq; 4 was prepared following the literature method
      • V V.
      • V G.
      Multicomponent synthesis of novel coelenterazine derivatives substituted at the C-3 position.
      ) in degassed ethanol was added 6N HCl (in water) and refluxed overnight. The reaction was concentrated and added 100 mL of 4 M HCl in dioxan and refluxed again overnight at 100 oC to deprotect the ethyl phosphonate groups. The reaction was concentrated on a rotary evaporator and the crude product was purified on a reverse-phase flash system using 120 g C18 column eluting with 5-100 % acetonitrile (0.1 % TFA) in water (0.1% water). The product fraction was pooled and concentrated to obtain the relatively pure product which was further purified on an HPLC system eluting with water/acetonitrile (modified with 0.1 % NH4OH) using the basic method. A small portion was further subjected to HPLC purification using an acidic method to obtain pure material of yellow CLZ-2P as a TFA salt. LC-MS Retention Time = 3.54 min (M+H)+ for C27H25N3O6P = 518.1. ; 1H NMR (400 MHz, dmso) δ 10.90 (brs, 1H), 9.73 (brs, 1H), 7.82 – 7.52 (m, 2H), 7.44 (d, J = 7.5 Hz, 2H), 7.34 – 7.17 (m, 5H), 6.95 – 6.88 (m, 2H), 6.88 – 6.80 (m, 2H), 4.30 (s, 2H), 4.07 – 3.96 (m, 4H); HRMS (ESI) m/z (M+H)+ calcd. for C27H25N3O6P = 518.1475, found 518.1560. (NMR spectrum for CLZ-2P, see Supplementary Figure 6)

      Data Availability

      Requests for materials and data supporting this study may be addressed to Brian Callahan ( [email protected] ).

      Conflict of Interest

      The authors are not aware of any conflict of interest

      Acknowledgements

      This research was supported (in part) by the Intramural Research Program of the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH) under project 1ZIATR000053 (J.I.), by the National Cancer Institute, Grant R01 CA206592 (BPC) and by National Institutes of General Medical Sciences, Grant R15GM143714 (JLG)

      REFERENCES

        • Rallis A.
        • Navarro J.A.
        • Rass M.
        • Hu A.
        • Birman S.
        • Schneuwly S.
        • Therond P.P.
        Hedgehog Signaling Modulates Glial Proteostasis and Lifespan.
        Cell Rep. 2020; 30: 2627-2643 e5
        • Lai K.
        • Kaspar B.K.
        • Gage F.H.
        • Schaffer D.V.
        Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo.
        Nat Neurosci. 2003; 6: 21-27
        • Liu Z.
        • Xu J.
        • He J.
        • Zheng Y.
        • Li H.
        • Lu Y.
        • Qian J.
        • Lin P.
        • Weber D.M.
        • Yang J.
        • Yi Q.
        A critical role of autocrine sonic hedgehog signaling in human CD138+ myeloma cell survival and drug resistance.
        Blood. 2014; 124: 2061-2071
        • Bissey P.A.
        • Mathot P.
        • Guix C.
        • Jasmin M.
        • Goddard I.
        • Costechareyre C.
        • Gadot N.
        • Delcros J.G.
        • Mali S.M.
        • Fasan R.
        • Arrigo A.P.
        • Dante R.
        • Ichim G.
        • Mehlen P.
        • Fombonne J.
        Blocking SHH/Patched Interaction Triggers Tumor Growth Inhibition through Patched-Induced Apoptosis.
        Cancer Res. 2020; 80: 1970-1980
        • Hong S.
        • Hu P.
        • Jang J.H.
        • Carrington B.
        • Sood R.
        • Berger S.I.
        • Roessler E.
        • Muenke M.
        Functional analysis of Sonic Hedgehog variants associated with holoprosencephaly in humans using a CRISPR/Cas9 zebrafish model.
        Hum Mutat. 2020; 41: 2155-2166
        • Roessler E.
        • El-Jaick K.B.
        • Dubourg C.
        • Velez J.I.
        • Solomon B.D.
        • Pineda-Alvarez D.E.
        • Lacbawan F.
        • Zhou N.
        • Ouspenskaia M.
        • Paulussen A.
        • Smeets H.J.
        • Hehr U.
        • Bendavid C.
        • Bale S.
        • Odent S.
        • David V.
        • Muenke M.
        The mutational spectrum of holoprosencephaly-associated changes within the SHH gene in humans predicts loss-of-function through either key structural alterations of the ligand or its altered synthesis.
        Hum Mutat. 2009; 30: E921-E935
        • Chen M.
        • Tanner M.
        • Levine A.C.
        • Levina E.
        • Ohouo P.
        • Buttyan R.
        Androgenic regulation of hedgehog signaling pathway components in prostate cancer cells.
        Cell cycle. 2009; 8: 149-157
        • Quaglio D.
        • Infante P.
        • Di Marcotullio L.
        • Botta B.
        • Mori M.
        Hedgehog signaling pathway inhibitors: an updated patent review (2015-present).
        Expert Opin Ther Pat. 2020; 30: 235-250
        • Jamieson C.
        • Martinelli G.
        • Papayannidis C.
        • Cortes J.E.
        Hedgehog Pathway Inhibitors: A New Therapeutic Class for the Treatment of Acute Myeloid Leukemia.
        Blood Cancer Discov. 2020; 1: 134-145
        • Petrova E.
        • Rios-Esteves J.
        • Ouerfelli O.
        • Glickman J.F.
        • Resh M.D.
        Inhibitors of Hedgehog acyltransferase block Sonic Hedgehog signaling.
        Nat Chem Biol. 2013; 9: 247-249
        • Lauth M.
        • Bergstrom A.
        • Shimokawa T.
        • Toftgard R.
        Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists.
        Proc Natl Acad Sci U S A. 2007; 104: 8455-8460
        • Rodgers U.R.
        • Lanyon-Hogg T.
        • Masumoto N.
        • Ritzefeld M.
        • Burke R.
        • Blagg J.
        • Magee A.I.
        • Tate E.W.
        Characterization of Hedgehog Acyltransferase Inhibitors Identifies a Small Molecule Probe for Hedgehog Signaling by Cancer Cells.
        ACS Chem Biol. 2016; 11: 3256-3262
        • Yauch R.L.
        • Dijkgraaf G.J.
        • Alicke B.
        • Januario T.
        • Ahn C.P.
        • Holcomb T.
        • Pujara K.
        • Stinson J.
        • Callahan C.A.
        • Tang T.
        • Bazan J.F.
        • Kan Z.
        • Seshagiri S.
        • Hann C.L.
        • Gould S.E.
        • Low J.A.
        • Rudin C.M.
        • de Sauvage F.J.
        Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma.
        Science. 2009; 326: 572-574
        • Dijkgraaf G.J.
        • Alicke B.
        • Weinmann L.
        • Januario T.
        • West K.
        • Modrusan Z.
        • Burdick D.
        • Goldsmith R.
        • Robarge K.
        • Sutherlin D.
        • Scales S.J.
        • Gould S.E.
        • Yauch R.L.
        • de Sauvage F.J.
        Small molecule inhibition of GDC-0449 refractory smoothened mutants and downstream mechanisms of drug resistance.
        Cancer Res. 2011; 71: 435-444
        • Robinson G.W.
        • Orr B.A.
        • Wu G.
        • Gururangan S.
        • Lin T.
        • Qaddoumi I.
        • Packer R.J.
        • Goldman S.
        • Prados M.D.
        • Desjardins A.
        • Chintagumpala M.
        • Takebe N.
        • Kaste S.C.
        • Rusch M.
        • Allen S.J.
        • Onar-Thomas A.
        • Stewart C.F.
        • Fouladi M.
        • Boyett J.M.
        • Gilbertson R.J.
        • Curran T.
        • Ellison D.W.
        • Gajjar A.
        Vismodegib Exerts Targeted Efficacy Against Recurrent Sonic Hedgehog-Subgroup Medulloblastoma: Results From Phase II Pediatric Brain Tumor Consortium Studies PBTC-025B and PBTC-032.
        J Clin Oncol. 2015; 33: 2646-2654
        • Belloni E.
        • Muenke M.
        • Roessler E.
        • Traverso G.
        • Siegel-Bartelt J.
        • Frumkin A.
        • Mitchell H.F.
        • Donis-Keller H.
        • Helms C.
        • Hing A.V.
        • Heng H.H.
        • Koop B.
        • Martindale D.
        • Rommens J.M.
        • Tsui L.C.
        • Scherer S.W.
        Identification of Sonic hedgehog as a candidate gene responsible for holoprosencephaly.
        Nat Genet. 1996; 14: 353-356
        • Bumcrot D.A.
        • Takada R.
        • McMahon A.P.
        Proteolytic processing yields two secreted forms of sonic hedgehog.
        Mol Cell Biol. 1995; 15: 2294-2303
        • Porter J.A.
        • Young K.E.
        • Beachy P.A.
        Cholesterol modification of hedgehog signaling proteins in animal development.
        Science. 1996; 274: 255-259
        • Callahan B.P.
        • Wang C.
        Hedgehog Cholesterolysis: Specialized Gatekeeper to Oncogenic Signaling.
        Cancers (Basel). 2015; 7: 2037-2053
        • Hall T.M.
        • Porter J.A.
        • Young K.E.
        • Koonin E.V.
        • Beachy P.A.
        • Leahy D.J.
        Crystal structure of a Hedgehog autoprocessing domain: homology between Hedgehog and self-splicing proteins.
        Cell. 1997; 91: 85-97
        • Perler F.B.
        Protein splicing of inteins and hedgehog autoproteolysis: structure, function, and evolution.
        Cell. 1998; 92: 1-4
        • Xie J.
        • Owen T.
        • Xia K.
        • Callahan B.
        • Wang C.
        A Single Aspartate Coordinates Two Catalytic Steps in Hedgehog Autoprocessing.
        J Am Chem Soc. 2016; 138: 10806-10809
        • Chen X.
        • Tukachinsky H.
        • Huang C.H.
        • Jao C.
        • Chu Y.R.
        • Tang H.Y.
        • Mueller B.
        • Schulman S.
        • Rapoport T.A.
        • Salic A.
        Processing and turnover of the Hedgehog protein in the endoplasmic reticulum.
        J Cell Biol. 2011; 192: 825-838
        • Huang C.H.
        • Hsiao H.T.
        • Chu Y.R.
        • Ye Y.
        • Chen X.
        Derlin2 protein facilitates HRD1-mediated retro-translocation of sonic hedgehog at the endoplasmic reticulum.
        J Biol Chem. 2013; 288: 25330-25339
        • Tang H.Y.
        • Huang C.H.
        • Zhuang Y.H.
        • Christianson J.C.
        • Chen X.
        EDEM2 and OS-9 are required for ER-associated degradation of non-glycosylated sonic hedgehog.
        PLoS One. 2014; 9e92164
        • Ciulla D.A.
        • Jorgensen M.T.
        • Giner J.L.
        • Callahan B.P.
        Chemical Bypass of General Base Catalysis in Hedgehog Protein Cholesterolysis Using a Hyper-Nucleophilic Substrate.
        J Am Chem Soc. 2018; 140: 916-918
        • Amakye D.
        • Jagani Z.
        • Dorsch M.
        Unraveling the therapeutic potential of the Hedgehog pathway in cancer.
        Nat Med. 2013; 19: 1410-1422
        • Hall M.P.
        • Unch J.
        • Binkowski B.F.
        • Valley M.P.
        • Butler B.L.
        • Wood M.G.
        • Otto P.
        • Zimmerman K.
        • Vidugiris G.
        • Machleidt T.
        • Robers M.B.
        • Benink H.A.
        • Eggers C.T.
        • Slater M.R.
        • Meisenheimer P.L.
        • Klaubert D.H.
        • Fan F.
        • Encell L.P.
        • Wood K.V.
        Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.
        ACS Chem Biol. 2012; 7: 1848-1857
        • Vincent S.
        • Thomas A.
        • Brasher B.
        • Benson J.D.
        Targeting of proteins to membranes through hedgehog auto-processing.
        Nat Biotechnol. 2003; 21: 936-940
        • Mozhdehi D.
        • Luginbuhl K.M.
        • Dzuricky M.
        • Costa S.A.
        • Xiong S.
        • Huang F.C.
        • Lewis M.M.
        • Zelenetz S.R.
        • Colby C.D.
        • Chilkoti A.
        Genetically Encoded Cholesterol-Modified Polypeptides.
        J Am Chem Soc. 2019; 141: 945-951
        • Traiffort E.
        • Dubourg C.
        • Faure H.
        • Rognan D.
        • Odent S.
        • Durou M.R.
        • David V.
        • Ruat M.
        Functional characterization of sonic hedgehog mutations associated with holoprosencephaly.
        J Biol Chem. 2004; 279: 42889-42897
        • Cooper M.K.
        • Porter J.A.
        • Young K.E.
        • Beachy P.A.
        Teratogen-mediated inhibition of target tissue response to Shh signaling.
        Science. 1998; 280: 1603-1607
        • Qiao Y.
        • Molina H.
        • Pandey A.
        • Zhang J.
        • Cole P.A.
        Chemical rescue of a mutant enzyme in living cells.
        Science. 2006; 311: 1293-1297
        • Lowry W.E.
        • Huang J.
        • Ma Y.C.
        • Ali S.
        • Wang D.
        • Williams D.M.
        • Okada M.
        • Cole P.A.
        • Huang X.Y.
        Csk, a critical link of g protein signals to actin cytoskeletal reorganization.
        Dev Cell. 2002; 2: 733-744
        • Marletta M.A.
        Raising enzymes from the dead and the secrets they can tell.
        ACS Chem Biol. 2006; 1: 73-74
        • Inglese J.
        • Johnson R.L.
        • Simeonov A.
        • Xia M.
        • Zheng W.
        • Austin C.P.
        • Auld D.S.
        High-throughput screening assays for the identification of chemical probes.
        Nat Chem Biol. 2007; 3: 466-479
        • He X.
        • Tan C.
        • Wang F.
        • Wang Y.
        • Zhou R.
        • Cui D.
        • You W.
        • Zhao H.
        • Ren J.
        • Feng B.
        Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.
        Nucleic Acids Res. 2016; 44: e85
        • Iannotti M.J.
        • MacArthur R.
        • Jones R.
        • Tao D.
        • Singec I.
        • Michael S.
        • Inglese J.
        Detecting Secretory Proteins by Acoustic Droplet Ejection in Multiplexed High-Throughput Applications.
        ACS Chem Biol. 2019; 14: 497-505
        • Lindberg E.
        • Mizukami S.
        • Ibata K.
        • Fukano T.
        • Miyawakide A.
        • Kikuchi K.
        Development of cell-impermeable coelenterazine derivatives.
        Chemical Science. 2013; 4: 4395-4400
        • Chang D.T.
        • Lopez A.
        • von Kessler D.P.
        • Chiang C.
        • Simandl B.K.
        • Zhao R.
        • Seldin M.F.
        • Fallon J.F.
        • Beachy P.A.
        Products, genetic linkage and limb patterning activity of a murine hedgehog gene.
        Development. 1994; 120: 3339-3353
        • Jiang S.Q.
        • Paulus H.
        A high-throughput, homogeneous, fluorescence polarization assay for inhibitors of hedgehog protein autoprocessing.
        J Biomol Screen. 2010; 15: 1082-1087
        • Owen T.S.
        • Ngoje G.
        • Lageman T.J.
        • Bordeau B.M.
        • Belfort M.
        • Callahan B.P.
        Forster resonance energy transfer-based cholesterolysis assay identifies a novel hedgehog inhibitor.
        Anal Biochem. 2015; 488: 1-5
        • Inglese J.
        • Auld D.S.
        • Jadhav A.
        • Johnson R.L.
        • Simeonov A.
        • Yasgar A.
        • Zheng W.
        • Austin C.P.
        Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries.
        Proc Natl Acad Sci U S A. 2006; 103: 11473-11478
        • Wagner A.G.
        • Stagnitta R.T.
        • Xu Z.
        • Pezzullo J.L.
        • Kandel N.
        • Giner J.L.
        • Covey D.F.
        • Wang C.
        • Callahan B.P.
        Nanomolar, Noncovalent Antagonism of Hedgehog Cholesterolysis: Exception to the "Irreversibility Rule" for Protein Autoprocessing Inhibition.
        Biochemistry. 2021;
        • Smith C.J.
        • Wagner A.G.
        • Stagnitta R.T.
        • Xu Z.
        • Pezzullo J.L.
        • Giner J.L.
        • Xie J.
        • Covey D.F.
        • Wang C.
        • Callahan B.P.
        Subverting Hedgehog Protein Autoprocessing by Chemical Induction of Paracatalysis.
        Biochemistry. 2020; 59: 736-741
        • Xie J.
        • Owen T.
        • Xia K.
        • Singh A.V.
        • Tou E.
        • Li L.
        • Arduini B.
        • Li H.
        • Wan L.Q.
        • Callahan B.
        • Wang C.
        Zinc inhibits Hedgehog autoprocessing: linking zinc deficiency with Hedgehog activation.
        J Biol Chem. 2015; 290: 11591-11600
        • Owen T.S.
        • Xie X.J.
        • Laraway B.
        • Ngoje G.
        • Wang C.
        • Callahan B.P.
        Active site targeting of hedgehog precursor protein with phenylarsine oxide.
        Chembiochem. 2015; 16: 55-58
        • V V.
        • V G.
        Multicomponent synthesis of novel coelenterazine derivatives substituted at the C-3 position.
        Tetrahedron. 2015; 71: 8781-8785