Effects of vanadate on the ATP content,ATPase activity and phosphate absorption capacity of maize roots

Although the sensitivity of the plasma membrane H⁺-ATPase to vanadate is well known, the metabolic response of plant cells to vanadate is less well characterised in vivo and its use as an inhibitor in whole plant experiments has had mixed success. Experiments with maize (Zea mays, L.) roots and with purified plasma membrane fractions from the same tissues showed that exposure to vanadate caused: (i) a reduction in the capacity for phosphate uptake; (ii) a reduction in the extractable ATPase activity from the tissue; and (iii) a significant increase in the ATP level. The measurements on the extractable ATPase activity and the ATP level showed that the effect of vanadate developed slowly, apparently reflecting the slow accumulation of intracellular vanadate. The marked effect of vanadate on the ATP level-exposure to 500 M vanadate for 5 h doubled the ATP content of the roots tips-indicates that there is no stringent control over the ATP level in the roots and that the plasma membrane H⁺-ATPase activity is likely to have a significant role in determining the ATP level under normal conditions.     

Imidazo[1,2-b]pyridazines: a potent and selective class of cyclin-dependent kinase inhibitors

Modification of imidazo[1,2-a]pyridine CDK inhibitors lead to identification of less lipophilic imidazo[1,2-b]pyridazine series of CDK inhibitors. Although several equivalent compounds from these two series have similar structure and show similar CDK activity, the SAR of the two series differs significantly. Protein inhibitor structure determination has confirmed differences in binding mode and given some understanding of these differences in SAR. Potent and selective imidazo[1,2-b]pyridazine inhibitors of CDK2 have been identified, which show >1 microM plasma levels following a 2mg/kg oral dose to mice.     

Structure-based design of protein tyrosine phosphatase-1B inhibitors

Using structure-based design, a new class of inhibitors of protein tyrosine phosphatase-1B (PTP1B) has been identified, which incorporate the 1,2,5-thiadiazolidin-3-one-1,1-dioxide template.     

Design and synthesis of a novel pyrrolidinyl pyrido pyrimidinone derivative as a potent inhibitor of PI3Kα and mTOR

Lead optimization efforts that employed structure base drug design and physicochemical property based optimization leading to the discovery of a novel series of 4-methylpyrido pyrimidinone (MPP) are discussed. Synthesis and profile of 1, a PI3Kα/mTOR dual inhibitor, is highlighted.     

SAR and inhibitor complex structure determination of a novel class of potent and specific Aurora kinase inhibitors

A novel series of 5-aminopyrimidinyl quinazolines has been developed from anilino-quinazoline 1, which was identified in a high throughput screen for Aurora A. Introduction of the pyrimidine ring and optimisation of the substituents both on this ring and at the C7 position of the quinazoline led to the discovery of compounds that are highly specific Aurora kinase inhibitors. Co-crystallisation of one of these inhibitors with a fragment of Aurora A shows the importance of the benzamido group in achieving selectivity.     

A novel small-molecule MRCK inhibitor blocks cancer cell invasion

Background

Dual oxidase maturation factor 1 (DUOXA1) has been associated with the maturation of the reactive oxygen species (ROS) producing enzyme, dual oxidase 1 (DUOX1) in the adult thyroid. However, ROS have also been implicated in the development of several tissues. We found that activated muscle satellite cells and primary myoblasts isolated from mice express robust levels of DUOXA1 and that its levels are altered as cells differentiate.

Results

To determine whether DUOXA1 levels affect muscle differentiation, we used an adenoviral construct (pCMV5-DUOXA1-GFP) to drive constitutive overexpression of this protein in primary myoblasts. High levels of DUOXA1 throughout myogenesis resulted in enhanced H₂O₂ production, fusion defects, reduced expression of early (myogenin) and late (myosin heavy chain) markers of differentiation, and elevated levels of apoptosis compared to control cells infected with an empty adenoviral vector (pCMV5-GFP). DUOXA1 knockdown (using a DUOXA1 shRNA construct) resulted in enhanced differentiation compared to cells subjected to a control shRNA, and subjecting DUOXA1 overexpressing cells to siRNAs targeting DUOX1 or apoptosis signal-regulating kinase 1 (ASK1) rescued the phenotype.

Conclusions

This study represents the first to demonstrate the importance of DUOXA1 in skeletal muscle myoblasts and that DUOXA1 overexpression in muscle stem cells induces apoptosis and inhibits differentiation through DUOX1 and ASK1.     

Structure-based design,synthesis and biological evaluation of a novel series of isoquinolone and pyrazolo[4,3-c]pyridine inhibitors of fascin 1 as potential anti-metastatic agents

Fascin is an actin binding and bundling protein that is not expressed in normal epithelial tissues but overexpressed in a variety of invasive epithelial tumors. It has a critical role in cancer cell metastasis by promoting cell migration and invasion. Here we report the crystal structures of fascin in complex with a series of novel and potent inhibitors. Structure-based elaboration of these compounds enabled the development of a series with nanomolar affinities for fascin, good physicochemical properties and the ability to inhibit fascin-mediated bundling of filamentous actin. These compounds provide promising starting points for fascin-targeted anti-metastatic therapies.     

Crystallographic studies of the anthrax lethal toxin

Anthrax lethal toxin comprises two proteins: protective antigen (PA; MW 83 kDa) and lethal factor (LF; MW 87 kDa). We have recently determined the crystal structure of the 735-residue PA in its monomeric and heptameric forms ( Petosa et al . 1997 ). It bears no resemblance to other bacterial toxins of known three-dimensional structure, and defines a new structural class which includes homologous toxins from other Gram-positive bacteria. We have proposed a model of membrane insertion in which the water-soluble heptamer undergoes a substantial pH-induced conformational change involving the creation of a 14-stranded β-barrel. Recent work by Collier's group ( Benson et al . 1998 ) lends strong support to our model of membrane insertion. 'Lethal factor' is the catalytic component of anthrax lethal toxin. It binds to the surface of the cell-bound PA heptamer and, following endocytosis and acidification of the endosome, translocates to the cytosol. We have made substantial progress towards an atomic resolution crystal structure of LF. Progress towards a structure of the 7:7 translocation complex between the PA heptamer and LF will also be discussed.     

Novel thienopyrrole glycogen phosphorylase inhibitors: synthesis, in vitro SAR and crystallographic studies

Two series of novel thienopyrrole inhibitors of recombinant human liver glycogen phosphorylase a (GPa) which are effective in reducing glucose output from rat hepatocytes are described. Representative compounds have been shown to bind at the dimer interface site of the rabbit muscle enzyme by X-ray crystallography.     

Fragment based discovery of a novel and selective PI3 kinase inhibitor

We report the use of fragment screening and fragment based drug design to develop a PI3γ kinase fragment hit into a lead. Initial fragment hits were discovered by high concentration biochemical screening, followed by a round of virtual screening to identify additional ligand efficient fragments. These were developed into potent and ligand efficient lead compounds using structure guided fragment growing and merging strategies. This led to a potent, selective, and cell permeable PI3γ kinase inhibitor with good metabolic stability that was useful as a preclinical tool compound.