A novel series of IKKβ inhibitors part II: description of a potent and pharmacologically active series of analogs

A novel series of (E)-1-((2-(1-methyl-1H-imidazol-5-yl) quinolin-4-yl) methylene) thiosemicarbazides was discovered as potent inhibitors of IKKβ. In this Letter we document our efforts at further optimization of this series, culminating in 2 with submicromolar potency in a HWB assay and efficacy in a CIA mouse model.     

Brassinin oxidase mediated transformation of the phytoalexin brassinin: structure of the elusive co-product, deuterium isotope effect and stereoselectivity

Brassinin oxidase, a fungal detoxifying enzyme that mediates the conversion of the phytoalexin brassinin into indole-3-carboxaldehyde, is the first enzyme described to date that catalyzes the transformation of a dithiocarbamate group into an aldehyde equivalent. Brassinin is an essential phytoalexin due to its antifungal activity and its role as biosynthetic precursor of other phytoalexins produced in plants of the family Brassicaceae (common name crucifer). In this report, the isolation, structure determination and synthesis of the elusive co-product of brassinin transformation by brassinin oxidase, S-methyl dithiocarbamate, the syntheses of dideuterated and (R) and (S) monodeuterated brassinins, kinetic analyses of isotope effects and chemical modifications of brassinin oxidase are described. The reaction of [1'-(2)H(2)]brassinin was found to be slowed by a kinetic isotope effect of 5.3 on the value of k(cat)/K(m). This result indicates that the hydride/hydrogen transfer step preceding brassinin transformation is rate determining in the overall reaction. In addition, the use of (R) and (S)-[1'-(2)H]brassinins as substrates indicated that the hydride/hydrogen transfer step is ca. 88% stereoselective for the pro-R hydrogen. A detailed chemical mechanism of the enzymatic transformation of brassinin is proposed.     

Spontaneous demonstration of counterclockwise right atrial activation following successful isthmus ablation

We describe an uncommon case of typical flutter with symptomatic sinus node dysfunction, in which a permanent junctional rhythm developed following ablation of the cavo-tricuspid isthmus. This rhythm activated the right atrium in counter clockwise manner thus providing spontaneous proof of unidirectional isthmus block, a phenomenon that is usually demonstrated by proximal coronary sinus pacing.     

Structure based prediction of functional sites with potential inhibitors to Nudix enzymes from disease causing microbes

The functional sites were predicted for Nudix enzymes from pathogenic microorganisms such as Streprococcus pneumonia (2B06) and Enterococcus faecalis (2AZW). Their structures are already determined, however, no data is reported about their functional sites, substrates and inhibitors. Therefore, we report prediction of functional sites in these Nudix enzymes via Geometric Invariant (GI) technique (Construct different geometries of peptides which remain unchanged). The GI method enumerated 2B06: RA57, EA58, EA61, EA62 and 2AZW: RA62, EA63, EA66, EA67 as putative functional sites in these Nudix enzymes. In addition, the substrate was predicted via Molecular docking (Docking of substrates against whole structure of Nudix enzymes). The substrate ADP-Ribose was docked with the Nudix enzymes, 2B06 (Docking energy -15.68 Kcal/mol) and 2AZW (Docking energy -10.86 Kcal/mol) with the higher affinity and the lower docking energy as compared to other substrates. The residues EA62 in 2B06 and RA62 in 2AZW make hydrogen bonds with the ADP-ribose. Furthermore, we screened 51 inhibitor compounds against structures of 2B06 and 2AZW. The inhibitor compounds AMPCPR and CID14258187 were docked well as compared to other compounds. The compound CID14258187 was also in agreement with Lipinski rule of 5 for drug likeness properties. Therefore, our findings of functional sites, substrates and inhibitors for these Nudix enzymes may help in structure based drug designing against Streprococcus pneumonia and Enterococcus faecalis.     

Simulating the mammalian blastocyst--molecular and mechanical interactions pattern the embryo

Mammalian embryogenesis is a dynamic process involving gene expression and mechanical forces between proliferating cells. The exact nature of these interactions, which determine the lineage patterning of the trophectoderm and endoderm tissues occurring in a highly regulated manner at precise periods during the embryonic development, is an area of debate. We have developed a computational modeling framework for studying this process, by which the combined effects of mechanical and genetic interactions are analyzed within the context of proliferating cells. At a purely mechanical level, we demonstrate that the perpendicular alignment of the animal-vegetal (a-v) and embryonic-abembryonic (eb-ab) axes is a result of minimizing the total elastic conformational energy of the entire collection of cells, which are constrained by the zona pellucida. The coupling of gene expression with the mechanics of cell movement is important for formation of both the trophectoderm and the endoderm. In studying the formation of the trophectoderm, we contrast and compare quantitatively two hypotheses: (1) The position determines gene expression, and (2) the gene expression determines the position. Our model, which couples gene expression with mechanics, suggests that differential adhesion between different cell types is a critical determinant in the robust endoderm formation. In addition to differential adhesion, two different testable hypotheses emerge when considering endoderm formation: (1) A directional force acts on certain cells and moves them into forming the endoderm layer, which separates the blastocoel and the cells of the inner cell mass (ICM). In this case the blastocoel simply acts as a static boundary. (2) The blastocoel dynamically applies pressure upon the cells in contact with it, such that cell segregation in the presence of differential adhesion leads to the endoderm formation. To our knowledge, this is the first attempt to combine cell-based spatial mechanical simulations with genetic networks to explain mammalian embryogenesis. Such a framework provides the means to test hypotheses in a controlled in silico environment.     

TC-PTP dephosphorylates the guanine nucleotide exchange factor C3G (RapGEF1) and negatively regulates differentiation of human neuroblastoma cells

The guanine nucleotide exchange factor, C3G (RapGEF1), functions in multiple signaling pathways involved in cell adhesion, proliferation, apoptosis and actin reorganization. C3G is regulated by tyrosine phosphorylation on Y504, known to be mediated by c-Abl and Src family kinases. In the present study we explored the possibility of cellular phospho-C3G (pC3G) being a substrate of the intracellular T-cell protein tyrosine phosphatase TC-PTP (PTPN2) using the human neuroblastoma cell line, IMR-32. In vivo and in vitro binding assays demonstrated interaction between C3G and TC-PTP. Interaction is mediated through the Crk-binding region of C3G and C-terminal noncatalytic residues of TC-PTP. C3G interacted better with a substrate trap mutant of TC48 and this complex formation was inhibited by vanadate. Endogenous pC3G colocalized with catalytically inactive mutant TC48 in the Golgi. Expression of TC48 abrogated pervanadate and c-Src induced phosphorylation of C3G without affecting total cellular phospho-tyrosine. Insulin-like growth factor treatment of c-Src expressing cells resulted in dephosphorylation of C3G dependent on the activity of endogenous TC48. TC48 expression inhibited forskolin induced tyrosine phosphorylation of C3G and neurite outgrowth in IMR-32 cells. Our results identify a novel Golgi localized substrate of TC48 and delineate a role for TC48 in dephosphorylation of substrates required during differentiation of human neuroblastoma cells.     

Designing nanoconjugates to effectively target pancreatic cancer cells in vitro and in vivo

Background

Pancreatic cancer is the fourth leading cause of cancer related deaths in America. Monoclonal antibodies are a viable treatment option for inhibiting cancer growth. Tumor specific drug delivery could be achieved utilizing these monoclonal antibodies as targeting agents. This type of designer therapeutic is evolving and with the use of gold nanoparticles it is a promising approach to selectively deliver chemotherapeutics to malignant cells.Gold nanoparticles (GNPs) are showing extreme promise in current medicinal research. GNPs have been shown to non-invasively kill tumor cells by hyperthermia using radiofrequency. They have also been implemented as early detection agents due to their unique X-ray contrast properties; success was revealed with clear delineation of blood capillaries in a preclinical model by CT (computer tomography). The fundamental parameters for intelligent design of nanoconjugates are on the forefront. The goal of this study is to define the necessary design parameters to successfully target pancreatic cancer cells.

Methodology/Principal Findings

The nanoconjugates described in this study were characterized with various physico-chemical techniques. We demonstrate that the number of cetuximab molecules (targeting agent) on a GNP, the hydrodynamic size of the nanoconjugates, available reactive surface area and the ability of the nanoconjugates to sequester EGFR (epidermal growth factor receptor), all play critical roles in effectively targeting tumor cells in vitro and in vivo in an orthotopic model of pancreatic cancer.

Conclusion

Our results suggest the specific targeting of tumor cells depends on a number of crucial components 1) targeting agent to nanoparticle ratio 2) availability of reactive surface area on the nanoparticle 3) ability of the nanoconjugate to bind the target and 4) hydrodynamic diameter of the nanoconjugate. We believe this study will help define the design parameters for formulating better strategies for specifically targeting tumors with nanoparticle conjugates.     

Natural images from the birthplace of the human eye

Here we introduce a database of calibrated natural images publicly available through an easy-to-use web interface. Using a Nikon D70 digital SLR camera, we acquired about six-megapixel images of Okavango Delta of Botswana, a tropical savanna habitat similar to where the human eye is thought to have evolved. Some sequences of images were captured unsystematically while following a baboon troop, while others were designed to vary a single parameter such as aperture, object distance, time of day or position on the horizon. Images are available in the raw RGB format and in grayscale. Images are also available in units relevant to the physiology of human cone photoreceptors, where pixel values represent the expected number of photoisomerizations per second for cones sensitive to long (L), medium (M) and short (S) wavelengths. This database is distributed under a Creative Commons Attribution-Noncommercial Unported license to facilitate research in computer vision, psychophysics of perception, and visual neuroscience.