Identification of the MACC1 transcriptional inhibitors via high-throughput screening (HTS).

<p>(A) Schematic representation of the reporter system used in the screening. The expression of the reporter firefly luciferase was regulated by the human MACC1 promoter (−992 to −18 bp upstream of the MACC1 transcriptional start site). (B) Diagrammatic representation of the HTS of HCT116-MACC1p-Luc cells with MACC1 promoter-driven luciferase expression. (C) Top 10 identified MACC1 promoter inhibitors identified from the HTS. (D–F) HCT116-MACC1p-Luc cells were treated with 10 two-fold serial dilutions of mevastatin (D), lovastatin (E), and rottlerin (F) for 24 h, starting with a 25 μM drug concentration. Luciferase activity was determined using steady glow luciferase reagent and normalized to untreated cells. Cell viability was measured independently by MTT assay. Results are shown as mean ± SEM of at least 2 independent experiments performed in triplicate.</p

The effect of mevastatin and lovastatin on MACC1 expression.

<p>(A–B) HCT116 cells were treated with increasing concentrations of mevastatin (<i>p</i> < 0.01, <i>p</i> < 0.001) (A) and lovastatin <i>(p</i> < 0.05, <i>p</i> < 0.001) (B) for 24 h. MACC1 mRNA expression and protein expression were determined by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. Treated samples are shown with black bars. MACC1 mRNA was normalized with G6PD and expressed as a percentage of solvent-treated cells. (C) HCT116 cells were treated with a single dose of 5 μM lovastatin for the time points indicated. MACC1 mRNA expression and protein expression were determined by qRT-PCR and western blot analysis, respectively (<i>p</i> < 0.001). (D) HCT116/vector and HCT116/MACC1 cells were treated with 5 μM lovastatin for 24 h, and MACC1 mRNA and protein levels were analyzed (<i>p</i> < 0.01). (E–G) SW48 (<i>p</i> < 0.001) (E), DLD-1 (<i>p</i> < 0.05, <i>p</i> < 0.01) (F), and SW620 (<i>p</i> < 0.01, <i>p</i> < 0.001) (G) cells were treated with 5 μM lovastatin for 24 h. MACC1 mRNA was analyzed by qRT-PCR, normalized with G6PD, and expressed as a percentage of solvent-treated cells. β-actin was used as the loading control for western blotting. Data represent mean ± SEM (<i>n</i> ≥ 2), *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

The effect of lovastatin on the binding of c-Jun and Sp1 to the MACC1 promoter.

<p>(A) An electrophoretic mobility shift assay (EMSA) was performed with equal amounts of nuclear extracts isolated from lovastatin- and solvent-treated cells. Lanes: (1) oligonucleotides only: 5′-biotin-labeled MACC1 promoter oligonucleotides flanking the binding sites specific for c-Jun (left) or Sp1 (right) without nuclear extract; (2) solvent control: nuclear extracts from solvent-treated HCT116 cells along with the respective labeled oligonucleotides; (3) lovastatin treatment: nuclear extracts from lovastatin-treated HCT116 cells along with the respective labeled oligonucleotides; (4) antibody: the DMSO-treated nuclear extracts were incubated with biotin-labeled oligonucleotides along with antibodies for c-Jun or Sp1; and (5) unlabeled oligos: a reaction with 100x molar excess of an unlabeled competitor sequence for indicating the specificity of the protein-DNA complexes; EMSA experiments have been performed at least 2 times, and a representative figure from each is shown. (B, D) Equal amounts of chromatin from solvent- and lovastatin-treated HCT116 cells were immunoprecipitated with antibodies for c-Jun (<i>p</i> < 0.05) (B) and Sp1 (D) and were quantified by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), using MACC1 promoter primers. The results were plotted as the percentage of input. (C, E) HCT116 cells were treated with 5 μM lovastatin for 24 h. Both mRNA expression and protein expression of c-Jun (C) and Sp1 (E) were analyzed. mRNA expression was normalized with G6PD and expressed as the percentage of the solvent-treated cells. β-actin was used as the loading control for western blotting. Data represent mean ± SEM (<i>n</i> = 2), *<i>p</i> < 0.05.</p

The effect of lovastatin on metastasis in mice.

<p>Severe combined immunodeficiency (SCID)-beige mice were intrasplenically transplanted with HCT116-CMVp-Luc cells and treated orally with 100 mg/kg lovastatin daily. Bioluminescence was measured by an intraperitoneal application of 150 mg/kg D-Luciferin and a sequence exposure of 20 s. (A) Acute toxicity was assessed in healthy animals treated with 100 mg/kg lovastatin. Body weight was measured daily for 10 d and is shown relative to day 0. (B, C) The lateral (B) and ventral signals (C) from tumors and metastases were monitored via bioluminescence imaging and quantified over time in solvent- and lovastatin-treated mice. (D, F) Representative pictures showing in vivo and ex vivo imaging of isolated organs from each group on day 26. All images are overlaid with the corresponding bright field pictures. Quantification of lateral (E) and ventral (<i>p</i> < 0.01) (G) signals was performed on day 26. All quantifications were performed using ImageJ software. (H) Human satellite DNA was quantified using quantitative polymerase chain reaction (qPCR) with equivalent amounts of genomic DNA obtained from the liver of each mouse. (I) MACC1 mRNA levels were determined from the livers using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and normalized to human G6PD (<i>p</i> < 0.05). Data represent mean ± SE (<i>n</i> = 9 animals/group), *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Molecular docking of transcriptional inhibitors to predict their mode of action.

<p>(A) 3D visualization of predicted drug binding to the DNA-binding domain (leucine zipper) of AP-1, viewed from different angles; mauve, rottlerin; green, mevastatin; and orange, lovastatin. (B) 2D visualization of predicted molecular interactions of the drugs with specific amino acid residues; the type of interaction is indicated by the color bar; mauve, rottlerin; green, mevastatin; and orange, lovastatin.</p

The effect of rottlerin on metastasis in the mice.

<p>Severe combined immunodeficiency (SCID)-beige mice were intrasplenically transplanted with HCT116-CMVp-Luc cells and treated orally with 100 mg/kg rottlerin daily. Bioluminescence was measured by an intraperitoneal application of 150 mg/kg D-Luciferin and a sequence exposure of 20 s. (A) Acute toxicity was assessed in healthy animals treated with 100 mg/kg rottlerin. Body weight was measured daily for 10 d and is shown relative to day 0. (B, C) The lateral (B) and ventral signals (C) from tumors and metastases were monitored via imaging and quantified over time in solvent- and rottlerin-treated mice. (D, F) Representative pictures showing in vivo and ex vivo imaging of mice and isolated organs from each group on day 24. All images are overlaid with the corresponding bright field pictures. Quantification of the lateral (<i>p</i> < 0.001) (E) and ventral (<i>p</i> < 0.05) (G) signals was performed on day 24. All quantifications are performed using ImageJ software. (H) Human satellite DNA was quantified using quantitative polymerase chain reaction (qPCR) with equivalent amounts of genomic DNA obtained from the liver of each mouse (<i>p</i> < 0.001). (I) MACC1 mRNA levels were determined from the livers using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and normalized to human G6PD (<i>p</i> < 0.05). Data represent mean ± SE (<i>n</i> = 9 animals/group), *<i>p</i> < 0.05, ***<i>p</i> < 0.001.</p

The effect of lovastatin on cell motility.

<p>(A) HCT116/vector and HCT116/MACC1 cells were treated with 5 μM lovastatin for 24 h, and migration was measured with the Boyden chamber assay. HCT116/MACC1 transfected with CMV promoter-driven MACC1 cDNA were used to revert the lovastatin effect. The migrated cells were stained with DAPI, and pictures were taken under a fluorescent microscope from 4 random fields per insert. The migrated cells were counted manually from those pictures and plotted in the bar graph. Data represent mean ± SEM (<i>n</i> = 2, <i>p</i> < 0.05). (B) The proliferation of lovastatin-treated versus nontreated cells was measured daily with MTT assays after the indicated time points. (C) The directed migration of lovastatin-treated HCT116 cells was analyzed by a wound healing assay. Microphotographs were taken 0 h, 24 h, and 48 h post treatment with 10x magnification. The assay was performed at least 2 times; 1 representative image is shown. (D) Wound size was analyzed using ImageJ and is expressed as the relative residual wound size compared to day 0. Control cells were treated with an equivalent amount of DMSO in all assays (<i>p</i> < 0.05).</p

The effect of rottlerin on MACC1 expression.

<p>(A–B) HCT116 cells were treated with increasing concentrations of rottlerin for 24 h (A) or a single dose of 2.5 μM rottlerin (B) for the time points indicated. MACC1 mRNA expression and protein expression were determined by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. Treated samples are shown with black bars. (C) HCT116/vector and HCT116/MACC1 cells were treated with 2.5 μM rottlerin for 24 h, and MACC1 mRNA and protein levels were analyzed (<i>p</i> < 0.01). (D–F) SW48 (D), DLD-1 (E), and SW620 (F) cells were treated with increasing concentrations of rottlerin for 24 h. MACC1 mRNA expression was analyzed by qRT-PCR. Samples with a 50% decrease in MACC1 mRNA levels are highlighted with black bars. MACC1 mRNA was normalized with G6PD and expressed as a percentage of solvent-treated cells (<i>p</i> < 0.001), whereas β-actin was used as loading control for western blotting. Data represent mean ± SEM (<i>n</i> ≥ 2), *<i>p</i> < 0.05, **<i>p</i> < 0.01 ***<i>p</i> < 0.001.</p

Probing Factor Xa Protein–Ligand Interactions: Accurate Free Energy Calculations and Experimental Validations of Two Series of High-Affinity Ligands

The accurate prediction of protein–ligand binding affinity belongs to one of the central goals in computer-based drug design. Molecular dynamics (MD)-based free energy calculations have become increasingly popular in this respect due to their accuracy and solid theoretical basis. Here, we present a combined study which encompasses experimental and computational studies on two series of factor Xa ligands, which enclose a broad chemical space including large modifications of the central scaffold. Using this integrated approach, we identified several new ligands with different heterocyclic scaffolds different from the previously identified indole-2-carboxamides that show superior or similar affinity. Furthermore, the so far underexplored terminal alkyne moiety proved to be a suitable non-classical bioisosteric replacement for the higher halogen−π aryl interactions. With this challenging example, we demonstrated the ability of the MD-based non-equilibrium free energy calculation approach for guiding crucial modifications in the lead optimization process, such as scaffold replacement and single-site modifications at molecular interaction hot spots

Structural Basis for Highly Selective Class II Alpha Phosphoinositide-3-Kinase Inhibition

Class II phosphoinositide-3-kinases (PI3Ks) play central roles in cell signaling, division, migration, and survival. Despite evidence that all PI3K class II isoforms serve unique cellular functions, the lack of isoform-selective inhibitors severely hampers the systematic investigation of their potential relevance as pharmacological targets. Here, we report the structural evaluation and molecular determinants for selective PI3K-C2α inhibition by a structure–activity relationship study based on a pteridinone scaffold, leading to the discovery of selective PI3K-C2α inhibitors called PITCOINs. Cocrystal structures and docking experiments supported the rationalization of the structural determinants essential for inhibitor activity and high selectivity. Profiling of PITCOINs in a panel of more than 118 diverse kinases showed no off-target kinase inhibition. Notably, by addressing a selectivity pocket, PITCOIN4 showed nanomolar inhibition of PI3K-C2α and >100-fold selectivity in a general kinase panel. Our study paves the way for the development of novel therapies for diseases related to PI3K-C2α function