Design, Palladium-Catalyzed Synthesis, and Biological Investigation of 2‑Substituted 3‑Aroylquinolin-4(1<i>H</i>)‑ones as Inhibitors of the Hedgehog Signaling Pathway

2-Substituted 3-aroylquinolin-4­(1<i>H</i>)-ones, prepared through a palladium-catalyzed carbonylative cyclization of <i>N</i>-(2-iodoaryl)­enaminones, proved to inhibit efficiently the Hedgehog pathway through direct antagonism of the wild-type and drug-resistant form of the Smoothened receptor. Notably, these compounds repressed the Hh-dependent growth events and the proliferation of tumor cells with aberrant activation of the Hh pathway, which plays a crucial role in development and tumorigenesis

Model of acetylation-dependent control of Gli activity.

<p>Following Hh/Ptch interaction, Smo triggers a signaling cascade leading to Gli2 deacetylation and to the inhibition of the βTrCP-regulated balance between Gli2R and full length Gli2 (Gli2A). Both events contributes to the early signal-dependent activation of the Hh pathway. Once activated, Gli2 promotes transcription of Gli1, whose activity is also regulated by Hh-induced HDAC1-mediated deacetylation, thus generating a positive feedback loop (late activation).</p

Acetylation of Gli transcription factors is not detectable in the External Granular Layer.

<p>Immunohistochemical staining of Gli1(upper-right), acetyl-Gli1(Lys518) (bottom-left), acetyl-GLi2(Lys757) (bottom-right), or Hematoxylin (upper-left) in a 6-day-old mouse cerebellum.</p

Gli2 acetylation prevents its promoter occupancy.

<p>(<b>A</b>) Cellular fractionation of NIH3T3 cells shows no difference in the localization of Myc tagged Gli2 wild type (WT), K575R and K575Q mutants. Purity of fractionation documented by tubulin (cytoplasmic, Cyto) and CREB (nuclear, Nu) staining. (<b>B</b>) Promoter occupancy of Gli2 is prevented by K757 acetylation. HEK293T cells were transfected with Myc tagged Gli2 wild type (WT), K757R, K757Q and Empty vectors and Chromatin Immunoprecipitation (ChIP) was carried out. Quantitative real-time PCR was performed using primers encompassing the Gli-BS of human <i>Ptch1</i> promoter (bottom, schematic representation). Results are indicated as fold difference, relative to Empty (pcDNA3) control. *p<0.05 WT vs Empty, **p<0.05 K757Q vs WT. Results are shown as the average ± SD of triplicate experiments (n = 4). Western blot analysis with anti Myc antibody showing equivalent amounts of Myc-Gli2 WT and mutants. Empty, pcDNA3.</p

Removal of Gli2 acetylation enhances Hh-dependent transcription.

<p>(<b>A</b>) K757 acetylation of wild type Myc tagged Gli2 and K757R mutant in HEK293T cells. Cells extract were immunoprecipitated with anti-Myc antibody and acetylation was detected by anti acetyl-Gli2(Lys757) specific antisera. Western blot analysis with anti Myc antibody showed equivalent amounts of Myc-Gli2. (<b>B</b>) Acetylation of endogenous Gli2 in NIH3T3 cells, treated with SAG or DMSO for 24 hours. Acetylation was detected with anti acetyl-Gli2(Lys757) specific antisera. (<b>C</b>) (Top) Luciferase assay in NIH3T3 cells transfected with 8× Gli-Luc reporter, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty. Results are shown as the average ± SD of triplicate experiments (n = 3). (Bottom) <i>Gli1</i> mRNA levels normalized with the housekeeping <i>HPRT</i> mRNA in NIH3T3 cells transfected with Myc-Gli2 WT and K757R mutant. (<b>D</b>) Transcriptional activity of Myc-Gli2 WT and K757R mutant in response to SAG treatment: <i>Ptch1</i> mRNA levels (QPCR), normalized with the housekeeping <i>GAPDH</i> mRNA in 24 hours SAG-treated NIH3T3 cells, transfected with Empty vector, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty, **p<0.05 vs DMSO; Results are shown as the average ± SD of triplicate experiments (n = 3).</p

p300 acetylates Gli2 at the conserved Lysine 757.

<p>(<b>A</b>) In vivo acetylation assay in HEK293T cells transfected with plasmids coding for Myc tagged Gli2 and the indicated HATs. Cell extracts were immunoprecipitated with anti-Myc antibody and acetylation was revealed with anti-acetyl lysine antisera. Western blot analysis with anti Myc antibody showed equivalent amounts of Myc-Gli2. Empty, pcDNA3. (<b>B</b>) Luciferase assay in HEK293T. Cells were transfected with 12× Gli-Luc and TK renilla reporter vectors, Gli2, p300, TIP60, pCAF where indicated. *p<0.01 vs Empty (pcDNA3). Results are shown as the average ± SD of triplicate experiments (n = 4). (<b>C</b>) (Top) ClustalW alignment of the region surrounding lysine 757 of Gli2 in different species. Identical, strongly conservative and weakly conservative aminoacids are indicated by asterisk, colon and dots respectively, according to ClustalW convention. (Bottom) In vivo acetylation assay in HEK293T cells transfected with Myc-Gli2 WT or K757R and p300 where indicated. Acetylation was detected as previously described. (<b>D</b>) (Top) Luciferase assay performed in HEK293T cells transfected with 12× Gli-Luc, TK renilla, Myc-Gli2 WT or the acetylation mimetic mutant Myc-Gli2 K757Q. *p<0.01 vs Empty; **p<0.05 vs WT. Results are shown as the average ± SD of triplicate experiments (n = 3). (Bottom) Western blot analysis of Myc-Gli2 WT and K757Q expression levels.</p

Superposition of the stapled peptides with Cul3.

<p>Superposition of the most representative conformer of Cul3^49-68EN (left panel; blue) and Cul3^49-68LA (right panel; yellow) respectively with the region (in red) encompassing residues Phe54-Leu66 of the crystal structure of Cul3 (PDB code 4EOZ).</p

Aminoacid sequences of all peptides.

<p>The replaced residues are highlighted in red. S₅ corresponds to the non-standard amino acid (S)-2-(4’-pentenyl) alanine.</p

Normalized Fluorescence Polarization data for Cul3^49-68LA as a function of [KCTD5^BTB].

<p>Peptide was plated at a final concentration of 1 μM, and the interaction with KCTD5^BTB was tested over a concentration range of 0.1 nM to 5 μM. Dissociation constants were calculated using nonlinear regression and are presented as mean ± standard error of triplicates.</p

Hα secondary chemical shift for Cul3^49-68 (a), Cul3^49-68LA (b) and Cul3^49-68EN (c) peptides.

<p>The red line indicates the average upfield shifts observed in peptides database for α-helix.</p