Neuroblastoma, an embryonal tumor of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer1. High-risk neuroblastomas, prevalent in the majority of patients, are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal2,3. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germline. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK cDNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed IL-3-dependent murine hematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to a small-molecule inhibitor of ALK, TAE6844. Furthermore, two human neuroblastoma cell lines harboring the F1174L mutation were sensitive to the inhibitor. Cytotoxicity was associated with increased levels of apoptosis as measured by TUNEL-labeling. shRNA-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumors and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.

Highly potent human glucagon receptor (hGluR) antagonists have been prepared employing both medicinal chemistry and targeted libraries based on modification of the core (proximal) dimethoxyphenyl group, the benzyl ether linkage, as well as the (distal) benzylic aryl group of the lead 2, 3-cyano-4-hydroxybenzoic acid (3,5-dimethoxy-4-isopropylbenzyloxybenzylidene)hydrazide. Electron-rich proximal aryl moieties such as mono- and dimethoxy benzenes, naphthalenes, and indoles were found to be active. The SAR was found to be quite insensitive regarding the linkage to the distal aryl group, since long and short as well as polar and apolar linkers gave highly potent compounds. The presence of a distal aryl group was not crucial for obtaining high binding affinity to the hGluR. In many cases, however, the affinity could be further optimized with substituted distal aryl groups. Representative compounds have been tested for in vitro metabolism, and structure-metabolism relationships are described. These efforts lead to the discovery of 74, NNC 25-2504, 3-cyano-4-hydroxybenzoic acid [1-(2,3,5,6-tetramethylbenzyl)-1H-indol-4-ylmethylene]hydrazide, with low in vitro metabolic turnover. 74 was a highly potent noncompetitive antagonist of the human glucagon receptor (IC(50) = 2.3 nM, K(B) = 760 pM) and of the isolated rat receptor (IC(50) = 430 pM, K(B) = 380 pM). Glucagon-stimulated glucose production from isolated primary rat hepatocytes was inhibited competitively by 74 (K(i) = 14 nM). This compound was orally available in dogs (F(po) = 15%) and was active in a glucagon-challenged rat model of hyperglucagonemia and hyperglycemia.

Aminoglycoside antibiotics target an internal RNA loop within the bacterial ribosomal decoding site. Here, we described the synthesis and SAR of novel 3,5-diamino-piperidine derivatives as aminoglycoside mimetics, and show they act as inhibitors of bacterial translation and growth. KeywordsAminoglycosides; Antibiotics; Translation inhibitors; 2-deoxy-streptamine; Ribosome; Decoding site; 3; 5-diamino-piperidine Bacterial resistance to antibiotics is on the rise and represents a global medical threat. In hospitals in the United States, approximately two million patients per year are infected. 1 The majority of these nosocomial pathogens are resistant to at least one antibiotic and result in about 90,000 deaths per year; a number that has increased 7-fold over the last decade. The recent and rapid spread of community acquired methicillin resistant Staphylococcus aureus further highlights the threat of resistance development and illustrates the need for new antibiotics that work by novel mechanisms. 2 Given the broad genetic and physiological diversity of bacterial pathogens and the need for empiric therapies that cover a broad panel of organisms, it is not surprising that discovery of new antibiotics has advanced slowly. Central to antibiotic discovery is identifying broadly validated targets. One such proven target is the bacterial ribosome, which is the target for a significant number of clinically important antibiotics that bind at the ribosomal RNA (rRNA). 3 Here, we expand on the description of a novel series of antibacterial compounds that target rRNA and blocks bacterial translation and growth. 4Three-dimensional structures of different aminoglycosides bound to the decoding site, or Asite, within the 16S rRNA have been determined by X-ray crystallography. 5 Importantly, these studies have shown that 2-deoxystreptamine (2-DOS), a conserved core scaffold among aminoglycosides, binds in a similar manner regardless of the 4,5-or 4,6-disubstitutions found in the neomycin or gentamicin families, respectively (Fig. 1) Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript groups of 2-DOS are predominantly involved in base recognition by forming conserved hydrogen bonds with A1493, G1494 and U1495 of the 16S rRNA. These interactions anchor the aminoglycoside scaffold within the A-site internal loop and displace residues A1492 and A1493 from the RNA interior. These two adenine residues act as a molecular switch that is involved in securing the fidelity of translation by interacting w...

We report the structure-guided discovery, synthesis, and initial characterization of 3,5-diamino-piperidinyl triazines (DAPT), a novel translation inhibitor class that targets bacterial rRNA and exhibits broad-spectrum antibacterial activity. DAPT compounds were designed as structural mimetics of aminoglycoside antibiotics which bind to the bacterial ribosomal decoding site and thereby interfere with translational fidelity. We found that DAPT compounds bind to oligonucleotide models of decoding-site RNA, inhibit translation in vitro, and induce translation misincorporation in vivo, in agreement with a mechanism of action at the ribosomal decoding site. The novel DAPT antibacterials inhibit growth of gram-positive and gram-negative bacteria, including the respiratory pathogen Pseudomonas aeruginosa, and display low toxicity to human cell lines. In a mouse protection model, an advanced DAPT compound demonstrated efficacy against an Escherichia coli infection at a 50% protective dose of 2.4 mg/kg of body weight by single-dose intravenous administration.