Gli1/ DNA interaction is a druggable target for Hedgehog‐dependent tumors
2014; Springer Nature; Volume: 34; Issue: 2 Linguagem: Inglês
10.15252/embj.201489213
ISSN1460-2075
AutoresPaola Infante, Mattia Mori, Romina Alfonsi, Francesca Ghirga, Federica Aiello, Sara Toscano, Cinzia Ingallina, Mariangela Siler, Danilo Cucchi, Agnese Pò, Evelina Miele, Davide D’Amico, Gianluca Canettieri, Enrico De Smaele, Elisabetta Ferretti, Isabella Screpanti, Gloria Uccello‐Barretta, Maurizio Botta, Bruno Botta, Alberto Gulino, Lucia Di Marcotullio,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle4 December 2014Open Access Source Data Gli1/DNA interaction is a druggable target for Hedgehog-dependent tumors Paola Infante Paola Infante Center for Life [email protected], Istituto Italiano di Tecnologia, Rome, Italy Search for more papers by this author Mattia Mori Mattia Mori Center for Life [email protected], Istituto Italiano di Tecnologia, Rome, Italy Search for more papers by this author Romina Alfonsi Romina Alfonsi Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Francesca Ghirga Francesca Ghirga Dipartimento di Chimica e Tecnologie del Farmaco, University La Sapienza, Rome, Italy Search for more papers by this author Federica Aiello Federica Aiello Department of Chemistry and Industrial Chemistry, University of Pisa, Pisa, Italy Search for more papers by this author Sara Toscano Sara Toscano Center for Life [email protected], Istituto Italiano di Tecnologia, Rome, Italy Search for more papers by this author Cinzia Ingallina Cinzia Ingallina Center for Life [email protected], Istituto Italiano di Tecnologia, Rome, Italy Search for more papers by this author Mariangela Siler Mariangela Siler Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Danilo Cucchi Danilo Cucchi Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Agnese Po Agnese Po Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Evelina Miele Evelina Miele Center for Life [email protected], Istituto Italiano di Tecnologia, Rome, Italy Search for more papers by this author Davide D'Amico Davide D'Amico Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Gianluca Canettieri Gianluca Canettieri Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Enrico De Smaele Enrico De Smaele Department of Experimental Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Elisabetta Ferretti Elisabetta Ferretti Department of Experimental Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Isabella Screpanti Isabella Screpanti Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Gloria Uccello Barretta Gloria Uccello Barretta Department of Chemistry and Industrial Chemistry, University of Pisa, Pisa, Italy Search for more papers by this author Maurizio Botta Maurizio Botta Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA, USA Search for more papers by this author Bruno Botta Corresponding Author Bruno Botta Dipartimento di Chimica e Tecnologie del Farmaco, University La Sapienza, Rome, Italy Search for more papers by this author Alberto Gulino Corresponding Author Alberto Gulino Center for Life [email protected], Istituto Italiano di Tecnologia, Rome, Italy Department of Molecular Medicine, University La Sapienza, Rome, Italy Istituto Pasteur, Fondazione Cenci-Bolognetti - University La Sapienza, Rome, Italy IRCCS Neuromed, Pozzilli, Italy Search for more papers by this author Lucia Di Marcotullio Corresponding Author Lucia Di Marcotullio Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Paola Infante Paola Infante Center for Life [email protected], Istituto Italiano di Tecnologia, Rome, Italy Search for more papers by this author Mattia Mori Mattia Mori Center for Life [email protected], Istituto Italiano di Tecnologia, Rome, Italy Search for more papers by this author Romina Alfonsi Romina Alfonsi Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Francesca Ghirga Francesca Ghirga Dipartimento di Chimica e Tecnologie del Farmaco, University La Sapienza, Rome, Italy Search for more papers by this author Federica Aiello Federica Aiello Department of Chemistry and Industrial Chemistry, University of Pisa, Pisa, Italy Search for more papers by this author Sara Toscano Sara Toscano Center for Life [email protected], Istituto Italiano di Tecnologia, Rome, Italy Search for more papers by this author Cinzia Ingallina Cinzia Ingallina Center for Life [email protected], Istituto Italiano di Tecnologia, Rome, Italy Search for more papers by this author Mariangela Siler Mariangela Siler Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Danilo Cucchi Danilo Cucchi Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Agnese Po Agnese Po Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Evelina Miele Evelina Miele Center for Life [email protected], Istituto Italiano di Tecnologia, Rome, Italy Search for more papers by this author Davide D'Amico Davide D'Amico Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Gianluca Canettieri Gianluca Canettieri Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Enrico De Smaele Enrico De Smaele Department of Experimental Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Elisabetta Ferretti Elisabetta Ferretti Department of Experimental Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Isabella Screpanti Isabella Screpanti Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Gloria Uccello Barretta Gloria Uccello Barretta Department of Chemistry and Industrial Chemistry, University of Pisa, Pisa, Italy Search for more papers by this author Maurizio Botta Maurizio Botta Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA, USA Search for more papers by this author Bruno Botta Corresponding Author Bruno Botta Dipartimento di Chimica e Tecnologie del Farmaco, University La Sapienza, Rome, Italy Search for more papers by this author Alberto Gulino Corresponding Author Alberto Gulino Center for Life [email protected], Istituto Italiano di Tecnologia, Rome, Italy Department of Molecular Medicine, University La Sapienza, Rome, Italy Istituto Pasteur, Fondazione Cenci-Bolognetti - University La Sapienza, Rome, Italy IRCCS Neuromed, Pozzilli, Italy Search for more papers by this author Lucia Di Marcotullio Corresponding Author Lucia Di Marcotullio Department of Molecular Medicine, University La Sapienza, Rome, Italy Search for more papers by this author Author Information Paola Infante1,‡, Mattia Mori1,‡, Romina Alfonsi2,‡, Francesca Ghirga3, Federica Aiello4, Sara Toscano1, Cinzia Ingallina1, Mariangela Siler2, Danilo Cucchi2, Agnese Po2, Evelina Miele1, Davide D'Amico2, Gianluca Canettieri2, Enrico De Smaele5, Elisabetta Ferretti5, Isabella Screpanti2, Gloria Uccello Barretta4, Maurizio Botta6,7, Bruno Botta 3, Alberto Gulino 1,2,8,9 and Lucia Di Marcotullio 2 1Center for Life [email protected], Istituto Italiano di Tecnologia, Rome, Italy 2Department of Molecular Medicine, University La Sapienza, Rome, Italy 3Dipartimento di Chimica e Tecnologie del Farmaco, University La Sapienza, Rome, Italy 4Department of Chemistry and Industrial Chemistry, University of Pisa, Pisa, Italy 5Department of Experimental Medicine, University La Sapienza, Rome, Italy 6Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy 7Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA, USA 8Istituto Pasteur, Fondazione Cenci-Bolognetti - University La Sapienza, Rome, Italy 9IRCCS Neuromed, Pozzilli, Italy ‡These authors contributed equally to this work *Corresponding author. Tel: +39 649255657; Fax: +39 649255660; E-mail: [email protected] or *Corresponding author. Tel: +39 649255129; Fax: +39 649255660; E-mail: [email protected] or *Corresponding author. Tel: +39 649912781; E-mail: [email protected] The EMBO Journal (2015)34:200-217https://doi.org/10.15252/embj.201489213 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Hedgehog signaling is essential for tissue development and stemness, and its deregulation has been observed in many tumors. Aberrant activation of Hedgehog signaling is the result of genetic mutations of pathway components or other Smo-dependent or independent mechanisms, all triggering the downstream effector Gli1. For this reason, understanding the poorly elucidated mechanism of Gli1-mediated transcription allows to identify novel molecules blocking the pathway at a downstream level, representing a critical goal in tumor biology. Here, we clarify the structural requirements of the pathway effector Gli1 for binding to DNA and identify Glabrescione B as the first small molecule binding to Gli1 zinc finger and impairing Gli1 activity by interfering with its interaction with DNA. Remarkably, as a consequence of its robust inhibitory effect on Gli1 activity, Glabrescione B inhibited the growth of Hedgehog-dependent tumor cells in vitro and in vivo as well as the self-renewal ability and clonogenicity of tumor-derived stem cells. The identification of the structural requirements of Gli1/DNA interaction highlights their relevance for pharmacologic interference of Gli signaling. Synopsis Structural features of the Gli1 zinc finger binding to DNA are explored to characterise Glabrescione B as novel and effective Hh-inhibitor in Gli1-dependent tumors. Binding of Gli1 zinc finger to DNA consensus sequence requires specific and previously uncharacterized residues. Glabrescione B, the first natural small molecule that binds these amino acids impairs Gli1 binding to DNA, inhibiting its transcriptional activity. Functionally, Glabrescione B shows antitumor efficacy both in vitro and in vivo. Introduction Inappropriate reactivation of the Hedgehog (Hh) developmental signaling pathway is responsible for the formation and progression of several human cancers through aberrant regulation of the functional properties of cancer stem cells (i.e. self-renewal, survival, metastatic spread, neoangiogenesis) (reviewed in Gulino et al, 2012; Amakye et al, 2013; Briscoe & Thèrond, 2013; Aberger & Ruiz I Altaba, 2014). Autocrine/paracrine Shh, Ihh or Dhh ligands bind to Patched (Ptch) receptor relieving its repressive activity on the seven-transmembrane protein Smoothened (Smo), which in turn activates the downstream transcription factors belonging to the Gli family. Gli proteins harbor a five zinc finger (ZF) region in which ZF4 and ZF5 domains bind target DNA in a sequence-specific way, whereas ZF1, ZF2 and ZF3 bind the phosphate backbone and possibly contributed to control binding stability and recruitment of co-regulatory factors (Kinzler & Vogelstein, 1990; Pavletich & Pabo, 1993). A C-terminal region is provided of transactivating function through modulation of chromatin remodeling induced by recruitment of TFIID TATA box-binding protein-associated factor TAFII31 (Yoon et al, 1998; Bosco-Clément et al, 2013) or HAT and HDAC (Canettieri et al, 2010; Malatesta et al, 2013), SWI-SNF5 (Jagani et al, 2010) and SWI/SNF-like Brg/Brm-associated factor (Zhan et al, 2011). In this way, Gli transcription factors behave as the final effectors for the control of specific oncogenic target genes (Aberger & Ruiz I Altaba, 2014). Although many Hh-driven human cancers involve upstream pathway activation (i.e. either loss-of-function Ptch1 or gain-of-function Smo mutations or Smo activation by loss of cAMP/PKA-mediated inhibitory Gα-GNAS tumor suppressor or ligand overproduction) (Goodrich et al, 1997; Yauch et al, 2009; He et al, 2014), several tumors harbor Smo-independent increased function of the downstream Gli effectors, due to high protein levels or activatory mechanisms (i.e. Gli gene amplification or epigenetically driven overexpression, mutation in or loss of heterozygosity of SuFu gene or a number of post-synthetic modifications such as decreased ubiquitination-mediated degradation or acetylation of the Gli proteins or increased PI3K/mTOR/S6K1 kinase-dependent phosphorylation) (Kinzler et al, 1987; Taylor et al, 2002; Dahlén et al, 2004; Di Marcotullio et al, 2006a, 2011; Canettieri et al, 2010; Wang et al, 2012; Mazzà et al, 2013; Tang et al, 2014). Notwithstanding, the mechanisms of regulation of Gli functions and the way it interacts with target DNA and controls transcription are still poorly understood. Small molecules have been reported to represent helpful tools to understand the mechanisms of modulation of Hh/Gli function at the level of Smo transducer and Gli effector. This allowed the development of several inhibitors targeting components of the pathway in order to control tumor growth. However, whereas several Smo antagonists are currently investigated in clinical trials (GDC-0449 has been recently approved by FDA), a few Gli inhibitors have been identified (Mas & Ruiz I Altaba, 2010; Coni et al, 2013a). Therefore, novel drugs targeting specific molecular steps underlying Gli function would be beneficial either for a wide spectrum of patients whose tumors have high Gli protein levels and/or activity. Furthermore, downstream activation of Gli function is frequently occurring with the appearance of resistance to Smo antagonists observed during therapy (Galimberti et al, 2012; Amakye et al, 2013). The lack of information on the structural and functional requirements of Gli1/DNA interaction accounts for the low number of small molecules inhibiting Gli. HPI-1 and HPI-4 have been shown to target the post-translational events of Gli processing/activation downstream of Smo, such as increase of the proteolytic cleavage of Gli2-FL to its repressor form Gli2-R or overall Gli1 degradation (Hyman et al, 2009). Arsenic trioxide (ATO) has been recently shown to prevent Gli2 localization to primary cilium, thus leading to its proteolytic degradation, while binding-dependent inactivation of Gli1 has not been yet characterized (Kim et al, 2010; Beauchamp et al, 2011). Similarly, GANT61 inhibits Gli1/DNA binding only in living cells, suggesting that it indirectly impairs its interaction with target gene promoters by as yet unelucidated mechanisms (Lauth et al, 2007). Based on the knowledge of the crystallographic structure of the zinc finger domain of Gli1 (Gli1ZF) in complex with DNA (Pavletich & Pabo, 1993), together with NMR studies as well as computational and experimental mutagenesis, we clarify here the structural requirements of Gli1/DNA interaction and identify Glabrescione B (GlaB), an isoflavone naturally found in the seeds of Derris glabrescens (Leguminosae), as a novel small molecule that binds Gli1ZF and interferes with its interaction with DNA. This small molecule turned out to be an efficient inhibitor of the growth of Hh/Gli-dependent tumors and cancer stem cells in vitro and in vivo, indicating that Gli/DNA interference is an appealing therapeutic strategy to control the heterogeneous molecular changes leading to Hh/Gli pathway activation in cancer. Results Structural and functional requirements for Gli1/DNA interaction To identify the molecular bases of the function of the Gli1/DNA complex, we established a computational screening protocol, based on the available X-ray structure of cobalt ion-coordinated Gli1ZF in complex with DNA (Pavletich & Pabo, 1993) that, although structurally relevant, does not provide by itself information on the energy of the system. Therefore, we investigated the conformational dynamics using the physiological zinc within the Zn-coordination system of each zinc finger by performing four independent replicas of molecular dynamics (MD) simulations. A representative Gli1ZF structure was extracted from MD trajectories (Fig 1A, Supplementary Movie S1) for further ligand design in silico. Figure 1. Structure-based analysis of Gli1/DNA complex Representative Gli1ZF/DNA structure extrapolated from MD trajectories. Gli1ZF is shown as blue cartoon, residues involved in binding to DNA (based on single point mutation study) are shown as magenta sticks, and Zn ions as gray spheres. Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning. The ∆∆G was calculated along MD trajectories as the difference between the ∆G of each Gli1ZF alanine mutant and Gli1ZF-WT. Results are shown as ∆∆G values in kcal/mol calculated by means of the MM-PBSA methods ± SEM. Effect of Gli1ZF mutants on Gli1-dependent transcriptional activation. Luciferase assay was performed in HEK293T cells transfected with 12XGliBS-Luc (GliBS, Gli binding site), pRL-TK Renilla (normalization control), Flag-Gli1 WT or the indicated Flag-Gli1 mutants. Data show the mean ± SD of three independent experiments. *P < 0.01; **P < 0.05 versus Gli1 WT. Western blot analysis of Flag-Gli1 WT or the indicated Flag-Gli1 mutant expression levels (bottom panel). Specific H-bond interactions between K350 of Gli1ZF and Gli1 binding site consensus, as predicted by MD. Gli1/DNA binding. Double-stranded oligonucleotide containing the canonical GliBS sequence (5′–TTGCCTACCTGGGTGGTCTCTCCACTT–3′) or mutated GliBS sequence used as control (5′–TTGCCTACCTCCCACTTCTCTCCACTT–3′) was used as probe (P) in EMSA experiments. The assay was performed using recombinant GST-Gli1ZF-WT (Gli1 zinc finger fragment: aa 242–424), GST-Gli1ZF-K350A and GST-Gli1ZF-K340A. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST-Gli1ZF-WT or GST-Gli1ZF mutants bound to the labeled GliBS probe/GliBS-free probe normalized to the amount of GST-Gli1ZF-WT/DNA binding (as described in 4). *P < 0.05 versus Gli1 WT. Source data are available online for this figure. Source Data for Figure 1 [embj201489213-SourceData-Fig1.pdf] Download figure Download PowerPoint Based on MD analysis and X-ray structure, the impact of serine and basic residues of ZF4 and ZF5 on the thermodynamic stability of the Gli1ZF/DNA adduct was evaluated by computational alanine scanning. The delta energy (ΔG) of binding of Gli1 mutants to DNA was calculated along MD trajectories (Fig 1B, Supplementary Table S1) and compared to the wild-type Gli1ZF (Gli1ZF-WT) (ΔΔG). The results show that the strongest ΔΔG contribution was given by basic residues involved in H-bonding DNA bases or in electrostatic interaction with the phosphate backbone of DNA. Indeed, mutation in silico of K340, K350, R354 in ZF4 and K360, K371, R380 and K381 of ZF5 strongly impaired the Gli1ZF ΔG of binding to DNA. To correlate these observations with transactivating function of Gli1, we performed a functional assay in HEK293T cells, transiently expressing ectopic Gli1 or different Gli1ZF mutants and a Gli-dependent luciferase reporter driven by a Gli-responsive consensus sequence. Mutations in K350 and R354 in ZF4 or R380 and K381 in ZF5 completely abrogated the transcriptional activity of Gli1, whereas K340, K371 and K360 did it to a lesser extent. Notably, a significant linear correlation was obtained (R2 = 0.6918) by comparing each theoretical ΔΔG value with the respective percentage of luciferase activity observed in cell assays. Mutagenesis studies suggested that the above residues may be involved in Gli1 binding to DNA (Fig 1C, Supplementary Table S1). We chose the K350A and K340A mutants, showing the highest and intermediate ΔΔG of binding, respectively (Supplementary Table S1), to test their direct interaction with DNA. An electrophoretic mobility shift assay (EMSA) was performed using equal amounts of recombinant GST-Gli1ZF-WT or GST-Gli1ZF-K350A or GST-Gli1ZF-K340A (Supplementary Fig S2) and a DNA probe containing Gli-responsive consensus DNA sequence or a mutated version unable to bind Gli1 (Fig 1D and E). Comparison with the strong DNA binding affinity of the recombinant GST-Gli1ZF-WT confirms that GST-Gli1ZF-K350A was unable to bind DNA, whereas GST-Gli1ZF-K340A did it, albeit to a significantly lower extent (Fig 1E, Supplementary Fig S2). Notably, these results correlate with the impaired and intermediate transcriptional activity of Gli1ZF-K350A and Gli1ZF-K340A mutants in cell-based assays, respectively (Fig 1C), and suggest that both K350 and K340 are involved in DNA binding and transcriptional activity, although with a different strength. Virtual library and transcriptional screening of Gli1 interacting small molecules To understand whether Gli1 binding to DNA and function could be regulated by small molecules, an in house library composed of more than 800 unique natural compounds was docked toward the MD representative Gli1ZF structure using the GOLD program (Supplementary Fig S3) (Verdonk et al, 2003). Literature data (Sheng et al, 2006) and results of the mutagenesis study were used to set up docking and analyze ligand poses, respectively. Indeed, only small molecules able to interact at least with one of the basic residues highlighted by the mutagenesis study were selected. The ligand ΔG of binding was then computed by means of the MM-GBSA method (Mori et al, 2011) and divided by the number of heavy atoms to provide the ligand efficiency (LE) as the final scoring parameter. We identified six molecules (three vismiones, GlaB, the chalcone V94 and the opioid alkaloid narceine) putatively behaving as potential Gli1 inhibitors (Supplementary Fig S4A). To investigate the functional activity of these compounds as specific modulators of Gli1, we used the Gli-dependent luciferase reporter screening assay. Whereas GlaB and Vismione E inhibited luciferase activity to a similar extent as GANT61 (a previously characterized Gli1 antagonist, Lauth et al, 2007), Vismione B only partially displayed inhibitory activity and other compounds were not active at all in this context (Fig 2A, Supplementary Fig S4B and C). Since vismiones have been shown to be quite chemically unstable in different conditions (Delle Monache, 1985), thus possibly limiting the identification of the bioactive specie, we focused further studies only on GlaB (Fig 2B). Figure 2. GlaB inhibits Hh signaling by impairing Gli1 function Inhibition of Gli1-induced transcription in transfected HEK293T cells. HEK293T cells were transfected with 12XGliBS-Luc and pRL-TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated with increasing concentrations of GlaB or GANT61. Treatment time was 24 h, and control cells were treated with DMSO only. GlaB chemical structure with numbering scheme for NMR analysis. Inhibition of Gli1-induced transcription in transfected Smo−/− MEF cells. Smo−/− MEF cells were transfected with 12XGliBS-Luc and pRL-TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated for 24 h with increasing concentrations of GlaB or DMSO only as control. The graphs show the Hh target gene expression levels in Ptch1−/− MEFs treated for 48 h with GlaB and DMSO as a control. mRNA levels were determined by quantitative real-time PCR (qRT–PCR) normalized to endogenous control (β2-microglobulin and HPRT). Pfkfb3 gene was used as a negative control. Representative model of Hh signaling hyperactivation: in Ptch1−/− MEFs, constitutive activation of Smo and consequently of Gli1; in SuFu−/− MEFs, release of Gli1 suppression. SuFu−/− MEFs were treated for 48 h with GlaB and DMSO as a control. Gli1 and Ptch1 mRNA levels were determined by qRT–PCR normalized to β2-microglobulin and HPRT expression. Pfkfb3 gene was used as a negative control. Promoter occupancy of Gli1 is prevented by GlaB treatment. MEF WT cells were transfected with Flag-tagged Gli1 or empty vectors, and chromatin immunoprecipitation (ChIP) was carried out. qRT–PCR was performed using primers encompassing the Gli-BS of mouse Ptch1 promoter (right, schematic representation). Results are indicated as fold difference, relative to empty (pcDNA3) control. Ptch1 mRNA expression levels (left panel) were determined by qRT–PCR in Daoy cells transfected with siRNA specific for Gli1 and Gli2 (siGli1/2) or a non-specific control siRNA (siCtr) and treated for 24 h with GlaB or DMSO as a control. (right panel) The graph shows Gli1 and Gli2 mRNA expression levels determined by qRT–PCR in Daoy cells transfected with siGli1/2 or siCtr. Results are expressed as fold repression relative to control, and data were normalized to GAPDH and HPRT expression. Data information: All data show the mean ± SD of three independent experiments. *P < 0.05 versus DMSO. Download figure Download PowerPoint Structural requirements for Gli1/GlaB interaction To investigate whether the Gli1 inhibitory activity of GlaB was associated to the direct interaction of this small molecule with the transcription factor, we monitored by NMR spectroscopy the GlaB proton mono-selective relaxation rates (Rms), which prove the slowing down of the small molecule motion upon binding to a receptor (Valensin et al, 1986; Neuhaus & Williamson, 1989). To this end, the protons H-1 and H-3 and the C-2 and C-4 methoxyl groups were chosen as probe for the interaction of ring A, proton H-11, H-12 and H-15 for ring B and proton H-8 for ring C (Table 1; Fig 2B). Table 1. Monoselective relaxation rates (Rms, s−1) of selected protons of GlaB (0.412 mM, 600 MHz, DMSO-d6, 25°C) and corresponding values of normalized relaxation rates (ΔR/Rf, where ΔR = Rms-Rf) in different mixtures GlaB/GST-Gli1ZF GlaB/GST-Gli1ZF K340A GlaB/GST-Gli1ZF K340A/K350A GlaB/GST Proton RING Rf(s−1) ΔR/Rf ΔR/Rf ΔR/Rf ΔR/Rf H-1 A 0.41 0 0 0.03 0.02 2-OMe A 1.20 0 0 0.05 0.06 H-3 A 0.63 0 0 0.05 0.10 4-OMe A 1.39 0 0 n.d.a 0 H-8 C 0.36 0.13 0.05 0.07 0 H-11 B 0.63 1.20 0.15 0.38 0.22 H-12 B 0.89 0.95 0.23 n.d.b 0.03 H-15 B 0.76 0.22 0.22 0.14 0.14 a Relaxation rate of 4-OMe protons was not determined because its signal was partially superimposed to that of the water. b Relaxation rate of proton H-12 was not measured because of the large linewidth. First, mono-selective relaxation rates of free GlaB (Rf) were measured to normalize the Rms detected in GlaB/protein mixtures (Table 1). In the presence of GST-Gli1ZF, a significant increase in Rms was observed for the vicinal protons H-11 and H-12 (ring B), and to a lesser extent H-15 (ring B) and H-8 (ring C) (Table 1; Supplementary Fig S5). Instead, the Rms of ring A protons were not perturbed by the presence of GST-Gli1ZF-WT and very likely were not involved in binding to Gli1ZF. To rule out any possible interference by the sole recombinant GST, we also monitored the Rms of GlaB in the GlaB/GST mixture, showing a weak and unspecific involvement of all rings A, B and C (Table 1; Supplementary Fig S5). Therefore, ring B was identified as the specific molecular determinant for the interaction between GlaB and GST-Gli1ZF. Although NMR analysis of O-prenyl groups was not too informative, because of the broad proton signals, they might be crucial for the correct positioning of GlaB ring B. Indeed, molecular docking studies showed that GlaB and Vismione B and E preferably bind within the same site at the interface between ZF4 and ZF5, with a noticeable shape and pharmacophoric overlapping (Supplementary Fig S6). The O-prenyl group at C-13 of GlaB is clearly overimposed to that of Vismione E, suggesting that a prenyl chain in this position may be relevant for Gli1 inhibition. GlaB derivatives without prenyl chains (Supplementary Fig S7) were also tested through the Gli-dependent luciferase reporter assay, but none of them was active, reinforcing that ring B prenyl chains may be important for GlaB biological activity. To investigate the role of Gli1 K340 and K350 residues, both involved in DNA binding and transcriptional function (Fig 1C–E), we monitored Rms of GlaB with the GST-Gli1ZF-K340A single mutant or the GST-Gli1ZF-K340A/K350A double mutant. Notably, the specific interaction of GlaB ring B was preserved in binding to the Gli1ZF-K340A, although with weaker local affinity (Table 1; Supplementary Fig S5). This suggests that GlaB may adopt a similar binding conformation toward Gli1ZF-WT and Gli1ZF-K340A. In contrast, the additional K350A mutation in the Gli1ZF-K340A/K350A double mutant completely affects the capability of GlaB ring B to interact with Gli1ZF, thus providing a GlaB proton pattern spanning non-specifically throughout rings A, B and C, as observed with the sole GST (Table 1, Supplementary Fig S5). In summary, results of NMR studies show that GlaB interacts directly with Gli1 and further emphasize the role of K340 and K350. The ring B and prenyl groups turned out to be key determinants for GlaB activity. GlaB inhibits Hh signaling by imparing Gli1/DNA binding and transcriptional activity Confirming the ability of GlaB to target Gli1, this compound significatively inhibited the luciferase activity driven by a Gli-responsive element reporter in Smo−/− MEF cells transfected with ectopic Gli1 (Fig 2C). Consistently, GlaB reduced the expression of several endoge
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