Search

Whereas recent clinical studies report metastatic melanoma survival rates high as 30-50%, many tumors remain nonresponsive or become resistant to current therapeutic strategies. Analyses of The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) data set suggests that a significant fraction of melanomas potentially harbor gain-of-function mutations in the gene that encodes for the ErbB4 receptor tyrosine kinase. In this work, a drug discovery strategy was developed that is based on the observation that the Q43L mutant of the naturally occurring ErbB4 agonist Neuregulin-2beta (NRG2β) functions as a partial agonist at ErbB4. NRG2β/Q43L stimulates tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and inhibits agonist-induced ErbB4-dependent cell proliferation. Compounds that exhibit these characteristics likely function as ErbB4 partial agonists, and as such hold promise as therapies for ErbB4-dependent melanomas. Consequently, three highly sensitive and reproducible (Z' > 0.5) screening assays were developed and deployed for the identification of small-molecule ErbB4 partial agonists. Six compounds were identified that stimulate ErbB4 phosphorylation, fail to stimulate ErbB4-dependent cell proliferation, and appear to selectively inhibit ErbB4-dependent cell proliferation. Whereas further characterization is needed to evaluate the full therapeutic potential of these molecules, this drug discovery platform establishes reliable and scalable approaches for the discovery of ErbB4 inhibitors.

Background and Purpose: Techniques to stratify subgroups of patients with asymptomatic carotid artery disease are urgently needed to guide decisions on optimal treatment. Reliance on estimates of % luminal stenosis has not been effective, perhaps because that approach entirely disregards potentially important information on the pathological process in the wall of the artery., Methods: Since plaque lipid is a key determinant of plaque behaviour we used a newly validated, high-sensitivity T2-mapping MR technique for a systematic survey of the quantity and distribution of plaque lipid in patients undergoing endarterectomy. Lipid percentage was quantified in 50 carotid endarterectomy patients. Lipid distribution was tested, using two imaging indices (contribution of the largest lipid deposit towards total lipid (LLD %) and a newly-developed LAI 'lipid aggregation index')., Results: The bifurcation contained maximal lipid volume. Lipid percentage was higher in symptomatic vs. asymptomatic patients with degree of stenosis (DS ≥ 50%) and in the total cohort (P = 0.013 and P = 0.005, respectively). Both LLD % and LAI was higher in symptomatic patients (P = 0.028 and P = 0.018, respectively), suggesting that for a given plaque lipid volume, coalesced deposits were more likely to be associated with symptomatic events. There was no correlation between plaque volume or lipid content and degree of luminal stenosis measured on ultrasound duplex (r = -0.09, P = 0.53 and r = -0.05, P = 0.75), respectively. However, there was a strong correlation in lipid between left and right carotid arteries (r = 0.5, P <0.0001, respectively)., Conclusions: Plaque lipid content and distribution is associated with symptomatic status of the carotid plaque. Importantly, plaque lipid content was not related to the degree of luminal stenosis assessed by ultrasound. Determination of plaque lipid content may prove useful for stratification of asymptomatic patients, including selection of optimal invasive treatments.

Mycobacterial tuberculosis (Mtb) is able to preserve its intrabacterial pH (pHIB) near neutrality in the acidic phagosomes of immunologically activated macrophages and to cause lethal pathology in immunocompetent mice. In contrast, when its ability to maintain pHIB homeostasis is genetically compromised, Mtb dies in acidic phagosomes and is attenuated in the mouse. Compounds that phenocopy the genetic disruption of Mtb's pHIB homeostasis could serve as starting points for drug development in their own right or through identification of their targets. A previously reported screen of a natural product library identified a phloroglucinol, agrimophol, that lowered Mtb's pHIB and killed Mtb at an acidic extrabacterial pH. Inability to identify agrimophol-resistant mutants of Mtb suggested that the compound may have more than one target. Given that polyphenolic compounds may undergo covalent reactions, we attempted an affinity-based method for target identification. The structure-activity relationship of synthetically tractable polyhydroxy diphenylmethane analogs with equivalent bioactivity informed the design of a bioactive agrimophol alkyne. After click-chemistry reaction with azido-biotin and capture on streptavidin, the biotinylated agrimophol analog pulled down the Mtb protein Rv3852, a predicted membrane protein that binds DNA in vitro. A ligand-protein interaction between agrimophol and recombinant Rv3852 was confirmed by isothermal calorimetry (ITC) and led to disruption of Rv3852's DNA binding function. However, genetic deletion of rv3852 in Mtb did not phenocopy the effect of agrimophol on Mtb, perhaps because of redundancy of its function.

Yersinia pestis forms a biofilm in the foregut of its flea vector that promotes transmission by flea bite. As in many bacteria, biofilm formation in Y. pestis is controlled by intracellular levels of the bacterial second messenger c-di-GMP. Two Y. pestis diguanylate cyclase (DGC) enzymes, encoded by hmsT and y3730, and one phosphodiesterase (PDE), encoded by hmsP, have been shown to control biofilm production in vitro via their opposing c-di-GMP synthesis and degradation activities, respectively. In this study, we provide further evidence that hmsT, hmsP, and y3730 are the only three genes involved in c-di-GMP metabolism in Y. pestis and evaluated the two DGCs for their comparative roles in biofilm formation in vitro and in the flea vector. As with HmsT, the DGC activity of Y3730 depended on a catalytic GGDEF domain, but the relative contribution of the two enzymes to the biofilm phenotype was influenced strongly by the environmental niche. Deletion of y3730 had a very minor effect on in vitro biofilm formation, but resulted in greatly reduced biofilm formation in the flea. In contrast, the predominant effect of hmsT was on in vitro biofilm formation. DGC activity was also required for the Hms-independent autoaggregation phenotype of Y. pestis, but was not required for virulence in a mouse model of bubonic plague. Our results confirm that only one PDE (HmsP) and two DGCs (HmsT and Y3730) control c-di-GMP levels in Y. pestis, indicate that hmsT and y3730 are regulated post-transcriptionally to differentially control biofilm formation in vitro and in the flea vector, and identify a second c-di-GMP-regulated phenotype in Y. pestis.

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.

The cuticle of Caenorhabditis elegans, a complex, multi-layered extracellular matrix, is a major interface between the animal and its environment. Biofilms produced by the bacterial genus Yersinia attach to the cuticle of the worm, providing an assay for surface characteristics. A C. elegans gene required for biofilm attachment, bah-1, encodes a protein containing the domain of unknown function DUF23. The DUF23 domain is found in 61 predicted proteins in C. elegans, which can be divided into three distinct phylogenetic clades. bah-1 is expressed in seam cells, which are among the hypodermal cells that synthesize the cuticle, and is regulated by a TGF-beta signaling pathway.