Resonance Raman spectroscopy of oxoiron(IV) porphyrin pi-cation radical and oxoiron(IV) hemes in peroxidase intermediates

The catalytic cycle intermediates of heme peroxidases, known as compounds I and II, have been of long standing interest as models for intermediates of heme proteins, such as the terminal oxidases and cytochrome P450 enzymes, and for non-heme iron enzymes as well. Reports of resonance Raman signals for compound I intermediates of the oxo-iron(IV) porphyrin pi-cation radical type have been sometimes contradictory due to complications arising from photolability, causing compound I signals to appear similar to those of compound II or other forms. However, studies of synthetic systems indicated that protein based compound I intermediates of the oxoiron(IV) porphyrin pi-cation radical type should exhibit vibrational signatures that are different from the non-radical forms. The compound I intermediates of horseradish peroxidase (HRP), and chloroperoxidase (CPO) from Caldariomyces fumago do in fact exhibit unique and characteristic vibrational spectra. The nature of the putative oxoiron(IV) bond in peroxidase intermediates has been under discussion in the recent literature, with suggestions that the Fe(IV)O unit might be better described as Fe(IV)-OH. The generally low Fe(IV)O stretching frequencies observed for proteins have been difficult to mimic in synthetic ferryl porphyrins via electron donation from trans axial ligands alone. Resonance Raman studies of iron-oxygen vibrations within protein species that are sensitive to pH, deuteration, and solvent oxygen exchange, indicate that hydrogen bonding to the oxoiron(IV) group within the protein environment contributes to substantial lowering of Fe(IV)O frequencies relative to those of synthetic model compounds.     

Imidazo[1,2-a]pyrazine diaryl ureas: Inhibitors of the receptor tyrosine kinase EphB4

Inhibition of receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs) has been validated by recently launched small molecules Sutent^® and Nexavar^®, both of which display activities against several angiogenesis-related RTKs. EphB4, a receptor tyrosine kinase (RTK) involved in the processes of embryogenesis and angiogenesis, has been shown to be aberrantly up regulated in many cancer types such as breast, lung, bladder and prostate. We propose that inhibition of EphB4 in addition to other validated RTKs would enhance the anti-angiogenic effect and ultimately result in more pronounced anti-cancer efficacy. Herein we report the discovery and SAR of a novel series of imidazo[1,2-a]pyrazine diarylureas that show nanomolar potency for the EphB4 receptor, in addition to potent activity against several other RTKs.     

Discovery of Candidate Disease Genes in ENU–Induced Mouse Mutants by Large-Scale Sequencing,Including a Splice-Site Mutation in Nucleoredoxin

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.     

Analysis of chromosomal insertion sites for Bacteroides Tn4555 and the role of TnpA

Tn4555, a mobilizable transposon carrying cefoxitin resistance, is directed to a preferred target site in the Bacteroides fragilis chromosome by a transposon-encoded targeting protein TnpA. In an effort to characterize target site selection for Tn4555, the existence of preferred target sites in other species of Bacteroides and in Escherichia coli was examined. For these analyses a Tn4555 mini element, pFD660, was transferred from E. coli donors to Bacteroides thetaiotaomicron or Bacteroides ovatus recipients and the resulting sites of insertion analyzed. A similar construct, pFD794 was used to determine insertion sites in E. coli, and preferred sites were found in all bacteria tested. Also the ability of TnpA to bind to various targets was examined in mobility shift assays. Although TnpA bound to all tested sequences, it displayed higher affinity for the target sites. The binding characteristics of TnpA and the lack of significant base sequence homology between targets suggested that secondary structure of the sites was important for TnpA binding. Circular permutation tests supported the idea that TnpA targets bent DNA.     

Toll-like receptor-7 agonist administered subcutaneously in a prolonged dosing schedule in heavily pretreated recurrent breast, ovarian, and cervix cancers

Background

The primary objective was to study the antitumor activity of prolonged subcutaneous dosing of systemic 852A, a Toll-like receptor-7 agonist (TLR-7), in recurrent breast, ovarian and cervix cancer. Secondary objectives included assessment of safety and immune system activation.

Methods

Adults with recurrent breast, ovarian or cervix cancer failing multiple therapies received 0.6 mg/m² of 852A subcutaneously twice weekly for 12 weeks. Doses increased by 0.2 mg/m²/week to a maximum of 1.2 mg/m². Serum was collected to assess immune activation.

Results

Fifteen patients enrolled: 10 ovarian, 2 cervix and 3 breast. Three completed all 24 injections. There were two grade 2 (decreased ejection fractions), nine grade 3 (1 cardiovascular, 1 anorexia, 3 dehydration, 2 infections, 2 renal) and two grade 4 (hepatic and troponin elevation) unanticipated toxicities. Cardiac toxicities included three cardiomyopathies (2 asymptomatic) and one stress-related non-ST elevated myocardial infarction. Five patients discontinued therapy due to possibly associated side effects. One who had stable disease (SD) following 24 doses received 17 additional doses. A cervix patient with SD following 24 doses received chemotherapy after progressing 3 months later, and remains disease free at 18 months. Immune activation, as evidenced by increased IP-10 and IL-1ra, was observed.

Conclusions

In this first human experience of a TLR-7 agonist delivered subcutaneously using a prolonged dosing schedule, 852A demonstrated sustained tolerability in some patients. Clinical benefit was modest, but immune activation was seen suggesting further study of antitumor applications is warranted. Because of cardiac toxicity; 852A should be used cautiously in heavily pretreated patients.     

Ligand Independence of the T618I Mutation in the Colony-stimulating Factor 3 Receptor (CSF3R) Protein Results from Loss of O-Linked Glycosylation and Increased Receptor Dimerization

Mutations in the CSF3 granulocyte colony-stimulating factor receptor CSF3R have recently been found in a large percentage of patients with chronic neutrophilic leukemia and, more rarely, in other types of leukemia. These CSF3R mutations fall into two distinct categories: membrane-proximal mutations and truncation mutations. Although both classes of mutation have exhibited the capacity for cellular transformation, several aspects of this transformation, including the kinetics, the requirement for ligand, and the dysregulation of downstream signaling pathways, have all been shown to be discrepant between the mutation types, suggesting distinct mechanisms of activation. CSF3R truncation mutations induce overexpression and ligand hypersensitivity of the receptor, likely because of the removal of motifs necessary for endocytosis and degradation. In contrast, little is known about the mechanism of activation of membrane-proximal mutations, which are much more commonly observed in chronic neutrophilic leukemia. In contrast with CSF3R truncation mutations, membrane-proximal mutations do not exhibit overexpression and are capable of signaling in the absence of ligand. We show that the Thr-615 and Thr-618 sites of membrane-proximal mutations are part of an O-linked glycosylation cluster. Mutation at these sites prevents O-glycosylation of CSF3R and increases receptor dimerization. This increased dimerization explains the ligand-independent activation of CSF3R membrane-proximal mutations. Cytokine receptor activation through loss of O-glycosylation represents a novel avenue of aberrant signaling. Finally, the combination of the CSF3R membrane proximal and truncation mutations, as has been reported in some patients, leads to enhanced cellular transformation when compared with either mutation alone, underscoring their distinct mechanisms of action.     

Molecular epidemiology of ESbetaL producing P. mirabilis strains from a long-term care and rehabilitation facility in Italy

We report the detection of multidrug resistant ESbetaL producing Proteus mirabilis isolates from a long-term care and rehabilitation facility (LTCRF) in Northern Italy. 53% of the collected P. mirabilis strains were ESbetaL producers. PCR and sequencing techniques confirmed the presence of the bla(TEM-92) and bla(CMY-16) resistance genes in 23/26 (88.5%) and 3/26 (11.5%) of the ESbetaL producers respectively. PFGE showed that the TEM-92 beta-lactamase producing isolates were not clonally related, indicating the presence of at least four different clonal lineages (A, B, C, D), whereas all the CMY-16 enzyme producers belonged in the same lineage. The bla(TEM-92) and bla(CYY-16) determinants were distributed in seven different wards, but in three of them they coexisted. Our results show that the most patients are co-colonized by ESbetaLs producing P. mirabilis strains at the time of admission to an LTCRF. An effective strategy to curtail the spread of ESbetaLs mediated resistance in LTCRFs could be to activate sourveillance programs to monitor routinely the entry of resistant bacteria.     

Synthesis and characterization of pyrrolidine derivatives as potent agonists of the human melanocortin-4 receptor

A series of trans-4-phenylpyrrolidine-3-carboxamides were synthesized and characterized as potent ligands of the human melanocortin-4 receptor. Interestingly, a pair of diastereoisomers 20f-1 and 20f-2 displayed potent functional agonist and antagonist activity, respectively. Thus, the 3S,4R-compound 20f-1 possessed a K(i) of 11nM and an EC(50) of 24nM, while its 3R,4S-isomer 20f-2 exhibited a K(i) of 8.6 and an IC(50) of 65nM. Both compounds were highly selective over other melanocortin receptor subtypes. The MC4R agonist 20f-1 also demonstrated efficacy in diet-induced obese rats.