Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe

The RAS–RAF–MEK–ERK pathway has been intensively studied in oncology, with RAS known to be mutated in ∼30% of all human cancers. The recent emergence of ERK1/2 inhibitors and their ongoing clinical investigation demands a better understanding of ERK1/2 behavior following small-molecule inhibition. Although fluorescent fusion proteins and fluorescent antibodies are well-established methods of visualizing proteins, we show that ERK1/2 can be visualized via a less-invasive approach based on a two-step process using inverse electron demand Diels–Alder cycloaddition. Our previously reported trans-cyclooctene-tagged covalent ERK1/2 inhibitor was used in a series of imaging experiments following a click reaction with a tetrazine-tagged fluorescent dye. Although limitations were encountered with this approach, endogenous ERK1/2 was successfully imaged in cells, and “on-target” staining was confirmed by over-expressing DUSP5, a nuclear ERK1/2 phosphatase that anchors ERK1/2 in the nucleus

A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase

Background: Examining essential biochemical pathways in Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite. Results: Introduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2. Conclusions: Allelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2. © 2016 The Author(s)

Homo-PROTACs:bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation

E3 ubiquitin ligases are key enzymes within the ubiquitin proteasome system which catalyze the ubiquitination of proteins, targeting them for proteasomal degradation. E3 ligases are gaining importance as targets to small molecules, both for direct inhibition and to be hijacked to induce the degradation of non-native neo-substrates using bivalent compounds known as PROTACs (for 'proteolysis-targeting chimeras'). We describe Homo-PROTACs as an approach to dimerize an E3 ligase to trigger its suicide-type chemical knockdown inside cells. We provide proof-of-concept of Homo-PROTACs using diverse molecules composed of two instances of a ligand for the von Hippel-Lindau (VHL) E3 ligase. The most active compound, CM11, dimerizes VHL with high avidity in vitro and induces potent, rapid and proteasome-dependent self-degradation of VHL in different cell lines, in a highly isoform-selective fashion and without triggering a hypoxic response. This approach offers a novel chemical probe for selective VHL knockdown, and demonstrates the potential for a new modality of chemical intervention on E3 ligases.Targeting the ubiquitin proteasome system to modulate protein homeostasis using small molecules has promising therapeutic potential. Here the authors describe Homo-PROTACS: small molecules that can induce the homo-dimerization of E3 ubiquitin ligases and cause their proteasome-dependent degradation

Dynamics of the corrosion for SAC305 solder alloy in salt environment

This study presents the corrosion dynamics of SAC305 solder alloy in two configurations (BGA solder balls components and solder material in globule) using a neutral salt spray test (NSS). SAC305 solder alloys were aged in different exposure durations ranging from 24 to 96 hours in a salt spray chamber with one salinity of 5 % NaCl at five temperatures ranging from 25°C to 45°C. Corrosion morphology, chemical composition and distribution of the elements in the corroded area and the corrosion product phases produced respectively after OM, SEM, EDX and XRD were also analyzed. The visible corrosion criterion defined on the OM and SEM results showed that the corroded areas are visible after 72h at 25°C, 60h at 30°C, 48h at 35°C, 36h at 40°C and only 24 h, at 45°C in case of BGAs solder balls components. The corroded area becomes larger at 96 hours. This indicates that temperature accelerates the corrosion mechanism. Moreover, according to the EDX result, tin (Sn), oxygen (O), and chloride ion (Cl-) were detected in the corroded area on solder balls. An activation energy (Ea) of 0.81 eV has been calculated. XRD measurements confirmed that a complex oxide chloride hydroxide (Sn3O(OH)2Cl2) was formed as a corrosion product on SAC globules. Finally, the kinetics of the failure mechanism of corrosion was investigated to refine the dynamic models of the degradation and overlay it on excessive stresses by simulations in order to predict lead-free solder alloys' lifetime

Exploring the Sm1-xCaxFe3-x tysonite solid solution as a solid-state electrolyte : relationship between structural features and F- ionic conductivity

Pure tysonite Sm1–xCaxF3–x solid solutions for 0.05 ≤ x ≤ 0.17 have been prepared by the solid-state route. For the first time, the partial Sm1–xCaxF3–x solid solution is investigated on the basis of structural features and ionic conductivity measurements. Powder X-ray diffraction Rietveld refinements show an unexpected decrease of the hexagonal unit cell volume related to the creation of fluorine vacancies. The local environment of F1, which is mainly responsible for the ionic conductivity, changes with the Ca content: the distortion of the F1(Sm,Ca)4 tetrahedral site decreases with the Ca content. Fluoride ion exchanges have been qualitatively probed on two Sm1–xCaxF3–x (x = 0.05 and x = 0.15) samples thanks to 19F magic angle spinning NMR experiments at various spinning frequencies and temperatures. At room temperature, the ionic conductivity decreases exponentially with the Ca content and the activation energy increases monotonously with the Ca content. The highest conductivity is found for the lowest Ca content or the smallest fluorine vacancy content stabilized in the Sm1–xCaxF3–x tysonite network corresponding to Sm0.95Ca0.05F2.95 (10–4 S·cm–1, Ea = 0.36 eV at room temperature). For this composition, the largest dispersions of F2–(Sm,Ca) and F3–(Sm,Ca) distances as well as (Sm,Ca)–F1–(Sm,Ca) angles are observed. The buckling of F2/F3 sheets around the z = 1/4 coordinate for low Ca content affects the large F1 tetrahedral site with the strongest distortion. The higher the buckling effect into the F2/F3 sheets, the higher the F1 local site distortion and the higher the ionic mobility and conductivity.Batteries tout solide à ions fluorur