The Small Molecule IMR-1 Inhibits the Notch Transcriptional Activation Complex to Suppress Tumorigenesis

In many cancers, aberrant Notch activity has been demonstrated to play a role in the initiation and maintenance of the neoplastic phenotype and in cancer stem cells, which may allude to its additional involvement in metastasis and resistance to therapy. Therefore, Notch is an exceedingly attractive therapeutic target in cancer, but the full range of potential targets within the pathway has been underexplored. To date, there are no small-molecule inhibitors that directly target the intracellular Notch pathway or the assembly of the transcriptional activation complex. Here, we describe an in vitro assay that quantitatively measures the assembly of the Notch transcriptional complex on DNA. Integrating this approach with computer-aided drug design, we explored potential ligand-binding sites and screened for compounds that could disrupt the assembly of the Notch transcriptional activation complex. We identified a small-molecule inhibitor, termed Inhibitor of Mastermind Recruitment-1 (IMR-1), that disrupted the recruitment of Mastermind-like 1 to the Notch transcriptional activation complex on chromatin, thereby attenuating Notch target gene transcription. Furthermore, IMR-1 inhibited the growth of Notch-dependent cell lines and significantly abrogated the growth of patient-derived tumor xenografts. Taken together, our findings suggest that a novel class of Notch inhibitors targeting the transcriptional activation complex may represent a new paradigm for Notch-based anticancer therapeutics, warranting further preclinical characterization. Cancer Res; 76(12); 3593-603. ©2016 AACR

Turning Defense into Offense: Defensin Mimetics as Novel Antibiotics Targeting Lipid II

<div><p>We have previously reported on the functional interaction of Lipid II with human alpha-defensins, a class of antimicrobial peptides. Lipid II is an essential precursor for bacterial cell wall biosynthesis and an ideal and validated target for natural antibiotic compounds. Using a combination of structural, functional and <i>in silico</i> analyses, we present here the molecular basis for defensin-Lipid II binding. Based on the complex of Lipid II with Human Neutrophil peptide-1, we could identify and characterize chemically diverse low-molecular weight compounds that mimic the interactions between HNP-1 and Lipid II. Lead compound BAS00127538 was further characterized structurally and functionally; it specifically interacts with the N-acetyl muramic acid moiety and isoprenyl tail of Lipid II, targets cell wall synthesis and was protective in an <i>in vivo</i> model for sepsis. For the first time, we have identified and characterized low molecular weight synthetic compounds that target Lipid II with high specificity and affinity. Optimization of these compounds may allow for their development as novel, next generation therapeutic agents for the treatment of Gram-positive pathogenic infections.</p></div

Crystal structure-based model of the HNP-1-Lipid II complex obtained with HADDOCK.

<p>(A) The HNP-1 dimer is shown in surface representation. (<b>B</b>) Residue Isoleucine 20 of HNP-1 monomer A interacts with Lysine-3 of the Lipid II pentapeptide, whereas Leucine residue 25 of HNP-1 monomer A interacts with D-Ala at position 4. (<b>C</b>) Residues Arginine15, Isoleucine 20 and Leucine25 of HNP-1 monomer B interact with γD-Glu-2 and the phosphate/N-acetyl muramic acid moiety of Lipid II. Critical residues in HNP-1 for the Lipid II interactions are shown in yellow and span the two monomers indicated in green and blue.</p

Characterization of BAS00127538.

<p>Chemical structure (left panel), bacterial killing (middle panel) and Lipid II binding (right panel) of defensin mimetic BAS00127538. Mimetic compound was 100% bactericidal at 0.244 µM against <i>S. aureus</i> and 7.8 µM against <i>E. coli</i>. Points of zero survival could not be plotted. (right panel) Representative sensorgrams of one out of three experiments of BAS00127538 binding to immobilized 3-Lipid II.</p

Residues involved in HNP-1 Lipid II contacts.

<p>Common three letters abbreviation is used for amino acids. D: amino acid in D-configuration. MurNac: N-acetyl Muramic acid. The identified contacts are based on an analysis of the top 4 docking models.</p

Analysis of the BAS00127538-lipid II complex by NMR.

<p>(Upper panel) Analysis of 2D TOCSY spectra collected at 800 MHz of the aromatic region of compound BAS00127538 alone (black) overlaid with spectra of compound bound to Lipid II (red) (Lower panel). 2D natural abunance ¹³C HSQC spectrum illustrating the interaction between Lipid II and the compound BAS00127538. BAS00127538 alone (black) is overlaid with a spectrum of compound bound to Lipid II (red). Spectra were collected on a Bruker 800 MHz Avance NMR spectrometer at 25 degrees. Chemical shift changes for Lipid II upon BAS00127538 compound binding suggest that the interaction is occuring at or near the MurNAc moeity of Lipid II.</p

Classification of lead defensin mimetics.

<p>N.D: Not Determinable by SPR. Bacterial killing: Concentration resulting in 100% killing after exposure of compound to bacteria for 30 min, determined by modified vCC assays <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003732#ppat.1003732-Ericksen1" target="_blank">[26]</a>. Binding to immobilized 3-Lipid II was analyzed by Surface Plasmon Resonance. C_50% equals compound concentration resulting in 50% cell survival measured by MTT assay following incubation for 24 h (Caco-2) or 4 h (Jurkat).</p