Biomedical subjects
Publications and source records attributed to James Trevillyan.
The synthesis and biological activities of rapamycin (I) analogs modified at the C-40 position are reported. Emphasis placed on compounds that potentially have an improved safety profile on account of their shorter in vivo half-life when compared with rapamycin.
Protein tyrosine phosphatases are important regulators of insulin signal transduction. Our studies have shown that in insulin resistant and diabetic ob/ob and db/db mice, reducing the levels of protein tyrosine phosphatase 1B (PTP1B) protein by treatment with a PTP1B antisense oligonucleotide resulted in improved insulin sensitivity and normalized plasma glucose levels. The mechanism by which PTP1B inhibition improves insulin sensitivity is not fully understood. We have used microarray analysis to compare gene expression changes in adipose tissue, liver and muscle of PTP1B antisense-treated ob/ob mice. Our results show that treatment with PTP1B antisense resulted in the downregulation of genes involved in lipogenesis in both fat and liver, and a downregulation of genes involved in adipocyte differentiation in fat, suggesting that PTP1B antisense acts through a different mechanism than thiazolidinedione (TZD) treatment. In summary, microarray results suggest that reduction of PTP1B may alleviate hyperglycemia and enhance insulin sensitivity by a different mechanism than TZD treatment.
In micro-arrayed compound screening (microARCS), an agarose gel is used as a reaction vessel that maintains humidity and compound location as well as being a handling system for reagent addition. Two or more agarose gels may be used to bring test compounds, targets, and reagents together, relying on the pore size of the gel matrix to regulate diffusion of reactants. It is in the microenvironment of the agarose matrix that all the components of an enzymatic reaction interact and result in inhibitable catalytic activity. In an effort to increase the throughput of microARCS-based screens, reduce the effort involved in manipulating agarose gels, and reduce costs, blotter paper was used rather than a second agarose gel to introduce a substrate to a gel containing a target enzyme. In this assay, the matrix of the blotter paper did not prevent the substrate from diffusing into the enzyme gel. The compound density of the microARCS format, the ease of manipulating sheets of paper for reagent addition, and a scheduled protocol for running multiple gels allowed for a throughput capacity of more than 200,000 tests per hour. A protease assay was developed and run in the microARCS format at a rate of 200,000 tests per hour using blotter paper to introduce the substrate. Picks in the primary screen were retested in the microARCS format at a density of 384 compounds per sheet. IC(50) values were confirmed in a 96-well plate format. The screen identified several small molecule inhibitors of the enzyme. The details of the screening format and the analysis of the hits from the screen are presented.
Participation of p38 mitogen-activated protein kinase (p38) in insulin-induced glucose uptake was suggested using pyridinylimidazole p38 inhibitors (e.g. SB203580). However, the role of p38 in insulin action remains controversial. We further test p38 participation in glucose uptake using a dominant-negative p38 mutant and two novel pharmacological p38 inhibitors related to but different from SB203580. We present the structures and activities of the azaazulene pharmacophores A291077 and A304000. p38 kinase activity was inhibited in vitro by A291077 and A304000 (IC(50) = 0.6 and 4.7 microm). At higher concentrations A291077 but not A304000 inhibited JNK2alpha (IC(50) = 3.5 microm). Pretreatment of 3T3-L1 adipocytes and L6 myotubes expressing GLUT4myc (L6-GLUT4myc myotubes) with A291077, A304000, SB202190, or SB203580 reduced insulin-stimulated glucose uptake by 50-60%, whereas chemical analogues inert toward p38 were ineffective. Expression of an inducible, dominant-negative p38 mutant in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. GLUT4 translocation to the cell surface, immunodetected on plasma membrane lawns of 3T3-L1 adipocytes or on intact L6-GLUT4myc myotubes, was not altered by chemical or molecular inhibition of p38. We propose that p38 contributes to enhancing GLUT4 activity, thereby increasing glucose uptake. In addition, the azaazulene class of inhibitors described will be useful to decipher cellular actions of p38 and JNK.