New dual inhibitors of EGFR and HER2 protein tyrosine kinases

A novel series of dual EGFR and HER2 inhibitors based on the pyrrolo[2,1-f][1,2,4]triazine nucleus is described. A general route toward their synthesis, which enables functionalization at multiple sites, has been developed. Biological evaluation in enzymatic and cell-based assays has identified a series of C-6 carbamates with potent biochemical and cellular activities.     

Arylphthalazines: identification of a new phthalazine chemotype as inhibitors of VEGFR kinase

A novel class of 4-arylamino-phthalazin-1-yl-benzamides is described as inhibitors of vascular endothelial growth factor receptor II (VEGFR-2). Several compounds display potent VEGFR-2 inhibitory activity with an IC50 as low as 0.078 microM in an HTRF enzymatic assay. These compounds are relatively selective against a small kinase panel.     

P2 pyridine N-oxide thrombin inhibitors: a novel peptidomimetic scaffold

In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P(1) benzylamide delivered the potent thrombin inhibitor 21 (K(i) = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels and half-life after oral dosing in the dog (C(max) = 2.6 microM, t(1/2) = 4.5 h).     

Synthesis and structure-activity relationships of 1,2,4-triazoles as a novel class of potent tubulin polymerization inhibitors

A novel triazole-containing chemical series was shown to inhibit tubulin polymerization and cause cell cycle arrest in A431 cancer cells with EC(50) values in the single digit nanomolar range. Binding experiments demonstrated that representative active compounds of this class compete with colchicine for its binding site on tubulin. The syntheses and structure-activity relationship studies for the triazole derivatives are described herein.     

A series of 5-(5,6)-dihydrouracil substituted 8-hydroxy-[1,6]naphthyridine-7-carboxylic acid 4-fluorobenzylamide inhibitors of HIV-1 integrase and viral replication in cells

Introduction of a 5,6-dihydrouracil functionality in the 5-position of N-(4-fluorobenzyl)-8-hydroxy-[1,6]naphthyridine-7-carboxamide 1 led to a series of highly active HIV-1 integrase inhibitors. These compounds displayed low nanomolar activity in inhibiting both the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 11 is a 150-fold more potent antiviral agent than 1, with a CIC(95) of 40 nM in the presence of human serum. It displays good pharmacokinetics when dosed in rats and dogs.     

Solid-phase synthesis and anti-infective activity of a combinatorial library based on the natural product anisomycin

The solid-phase synthesis of a library based on the natural product anisomycin is described. The resulting library was tested against a panel of bacterial and fungal targets, and active compounds were identified in a Staphylococcus aureus whole-cell assay and an efflux-deficient fungal whole-cell assay.     

Design and synthesis of potent pyridazine inhibitors of p38 MAP kinase

Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition of LPS-induced TNF-alpha production in mice and exhibited good efficacy in the rat collagen induced arthritis model.     

Release of ADP from the catalytic subunit of protein kinase A: a molecular dynamics simulation study

Substrate phosphorylation by cAMP-dependent-protein kinase A (protein kinase A, PKA) has been studied extensively. Phosphoryl transfer was found to be fast, whereas ADP release was found to be the slow, rate-limiting step. There is also evidence that ADP release may be preceded by a partially rate-limiting conformational change. However, the atomic details of the conformational change and the mode of ADP release are difficult to obtain experimentally. In this work, we studied ADP release from PKA by carrying out molecular dynamics simulations with different pulling forces applied to the ligand. The detailed ADP release pathway and the associated conformational changes were analyzed. The ADP release process was found to involve a swinging motion with the phosphate of ADP anchored to the Gly-rich loop, so that the more buried adenine base and ribose ring came out before the phosphate. In contrast to the common belief that a hinge-bending motion was responsible for the opening of the ligand-binding cleft, our simulations showed that the small lobe exhibited a large amplitude "rocking" motion when the ligand came out. The largest conformational change of the protein was observed at about the first quarter time point along the release pathway. Two prominent intermediate states were observed in the release process.     

Expression of human peripheral cannabinoid receptor for structural studies

Human peripheral-type cannabinoid receptor (CB2) was expressed in Escherichia coli as a fusion with the maltose-binding protein, thioredoxin, and a deca-histidine tag. Functional activity and structural integrity of the receptor in bacterial protoplast membranes was confirmed by extensive binding studies with a variety of natural and synthetic cannabinoid ligands. E. coli membranes expressing CB2 also activated cognate G-proteins in an in vitro coupled assay. Detergent-solubilized receptor was purified to 80%-90% homogeneity by affinity chromatography followed by ion-exchange chromatography. By high-resolution NMR on the receptor in DPC micelles, it was determined that purified CB2 forms 1:1 complexes with the ligands CP-55,940 and anandamide. The receptor was successfully reconstituted into phosphatidylcholine bilayers and the membranes were deposited into a porous substrate as tubular lipid bilayers for structural studies by NMR and scattering techniques.     

Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina

Cellular mechanisms underlying the precision by which neurons target their synaptic partners have largely been determined based on the study of projection neurons. By contrast, little is known about how interneurons establish their local connections in vivo. Here, we investigated how developing amacrine interneurons selectively innervate the appropriate region of the synaptic neuropil in the inner retina, the inner plexiform layer (IPL). Increases (ON) and decreases (OFF) in light intensity are processed by circuits that are structurally confined to separate ON and OFF synaptic sublaminae within the IPL. Using transgenic zebrafish in which the majority of amacrine cells express fluorescent protein, we determined that the earliest amacrine-derived neuritic plexus formed between two cell populations whose somata, at maturity, resided on opposite sides of this plexus. When we followed the behavior of individual amacrine cells over time, we discovered that they exhibited distinct patterns of structural dynamics at different stages of development. During cellular migration, amacrine cells exhibited an exuberant outgrowth of neurites that was undirected. Upon reaching the forming IPL, neurites extending towards the ganglion cell layer were relatively more stable. Importantly, when an arbor first formed, it preferentially ramified in either the inner or outer IPL corresponding to the future ON and OFF sublaminae, and maintained this stratification pattern. The specificity by which ON and OFF amacrine interneurons innervate their respective sublaminae in the IPL contrasts with that observed for projection neurons in the retina and elsewhere in the central nervous system.