Dual spectral phase and diffraction angle compensation of a broadband AOM 4-f pulse-shaper for ultrafast spectroscopy.

AOM-based pulse shaping as a method has been shown to provide many advantages in the field of ultrafast spectroscopy, in particular for the creation of phase matched pulse pairs for two-dimensional IR and electronic spectroscopy. In this paper we demonstrate the capabilities of a quartz-based AOM pulse-shaper to provide fine control over the phase and spatial dispersion of ultrafast supercontinuum pulses. We show that by using the Bragg condition, we can define a mask function for our AOM such that the angle of diffraction is constant for all frequencies. By summing all the contributions to spectral phase due to normal and anomalous dispersion of our optical components, and taking into account the intrinsic frequency dependent phase as a result of the acoustic sine wave propagating through the AOM, we can determine an optimal mask function that meets the Bragg condition for all frequencies, and generates compressed (∼50 fs) supercontinuum pulses.

Co-expression of protein phosphatases in insect cells affects phosphorylation status and expression levels of proteins.

The activity of kinases is regulated by phosphorylation on Ser, Thr or Tyr residues within the activation loop. The ability to produce these enzymes recombinantly with a specific phosphorylation status is essential in order to understand structure and function. In this paper we describe a screening approach to co-express different phosphatases together with a kinase in the baculovirus expression system. This enabled the testing of different phosphatases as well as different levels of both phosphatase and kinase by varying the multiplicity of infection (MOI) of the different baculoviruses. This approach translated well to a larger scale. An unexpected observation was that co-expression of the phosphatase could have profound effects on expression levels even of heterologous target proteins that would not be a substrate for the phosphatase. This was most apparent with lambda phosphatase, an enzyme that removes phosphorylation from Ser and Thr residues, where expression was almost completely abolished for all proteins, even at modest MOIs. The effect of lambda phosphatase was observed irrespective of whether co-expression was from two separate baculoviruses or from two genes on the same vector. The effect was shown to be due, in part at least, to a decrease in transcription.

Discovery of novel benzylidene-1,3-thiazolidine-2,4-diones as potent and selective inhibitors of the PIM-1, PIM-2, and PIM-3 protein kinases.

Novel substituted benzylidene-1,3-thiazolidine-2,4-diones (TZDs) have been identified as potent and highly selective inhibitors of the PIM kinases. The synthesis and SAR of these compounds are described, along with X-ray crystallographic, anti-proliferative, and selectivity data.

Inhibitors of the tyrosine kinase EphB4. Part 3: identification of non-benzodioxole-based kinase inhibitors.

Starting from the initial bis-anilinopyrimidine 1, good potency against EphB4 was retained when benzodioxole at C-4 was replaced by an indazole. The key interactions of the indazole with the protein were characterised by crystallographic studies. Further optimisation led to compound 20, a potent inhibitor of the EphB4 and Src kinases with good pharmacokinetics in various preclinical species and high fraction unbound in plasma. Compound 20 may be used as a tool for evaluating the potential of EphB4 kinase inhibitors in vivo.

Recurrent MLK4 Loss-of-Function Mutations Suppress JNK Signaling to Promote Colon Tumorigenesis.

MLK4 is a member of the mixed-lineage family of kinases that regulate the JNK, p38, and ERK kinase signaling pathways. MLK4 mutations have been identified in various human cancers, including frequently in colorectal cancer, where their function and pathobiological importance have been uncertain. In this study, we assessed the functional consequences of MLK4 mutations in colon tumorigenesis. Biochemical data indicated that a majority of MLK4 mutations are loss-of-function (LOF) mutations that can exert dominant-negative effects. In seeking to understand the abrogated activity of these mutants, we elucidated a new MLK4 catalytic domain structure. To determine whether MLK4 is required to maintain tumorigenic phenotypes, we reconstituted its signaling axis in colon cancer cells harboring MLK4-inactivating mutations. We found that restoring MLK4 activity reduced cell viability, proliferation, and colony formation in vitro and delayed tumor growth in vivo. Mechanistic investigations established that restoring the function of MLK4 selectively induced the JNK pathway and its downstream targets, cJUN, ATF3, and the cyclin-dependent kinase inhibitors CDKN1A and CDKN2B. Our work indicates that MLK4 is a novel tumor-suppressing kinase harboring frequent LOF mutations that lead to diminished signaling in the JNK pathway and enhanced proliferation in colon cancer.

Protein transduction domains: are they delivering?

Early studies with protein transduction domains (PTDs), such as those derived from Tat and Drosophila Antennapedia, showed rapid, receptor- and endosomal-independent uptake of conjugated biological tools into all cell types. However, recent mechanistic studies suggest that these observations were artefacts of the positively charged nature of PTDs and that uptake is instead via electrostatic binding to the plasma membrane and subsequent endocytosis. Given these observations, we assess the future utility of PTDs for in vitro and in vivo cellular delivery.

Structural basis of substrate methylation and inhibition of SMYD2.

Protein lysine methyltransferases are important regulators of epigenetic signaling. These enzymes catalyze the transfer of donor methyl groups from S-adenosylmethionine to specific acceptor lysines on histones, leading to changes in chromatin structure and transcriptional regulation. These enzymes also methylate nonhistone protein substrates, revealing an additional mechanism to regulate cellular physiology. The oncogenic protein SMYD2 represses the functional activities of the tumor suppressor proteins p53 and Rb, making it an attractive drug target. Here we report the discovery of AZ505, a potent and selective inhibitor of SMYD2 that was identified from a high throughput chemical screen. We also present the crystal structures of SMYD2 with p53 substrate and product peptides, and notably, in complex with AZ505. This substrate competitive inhibitor is bound in the peptide binding groove of SMYD2. These results have implications for the development of SMYD2 inhibitors, and indicate the potential for developing novel therapies targeting this target class.

Discovery of 5-chloro-N2-[(1S)-1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(5-methyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (AZD1480) as a novel inhibitor of the Jak/Stat pathway.

The myeloproliferative neoplasms, polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis are a heterogeneous but related group of hematological malignancies characterized by clonal expansion of one or more myeloid lineages. The discovery of the Jak2 V617F gain of function mutation highlighted Jak2 as a potential therapeutic target in the MPNs. Herein, we disclose the discovery of a series of pyrazol-3-yl pyrimidin-4-amines and the identification of 9e (AZD1480) as a potent Jak2 inhibitor. 9e inhibits signaling and proliferation of Jak2 V617F cell lines in vitro, demonstrates in vivo efficacy in a TEL-Jak2 model, has excellent physical properties and preclinical pharmacokinetics, and is currently being evaluated in Phase I clinical trials.