2,6-Naphthyridines as potent and selective inhibitors of the novel protein kinase C isozymes.

The present study describes a novel series of ATP-competitive PKC inhibitors based on the 2,6-naphthyridine template. Example compounds potently inhibit the novel Protein Kinase C (PKC) isotypes δ, ε, η, θ (in particular PKCε/η, and display a 10-100-fold selectivity over the classical PKC isotypes. The prototype compound 11 was found to inhibit PKCθ-dependent pathways in vitro and in vivo. In vitro, a-CD3/a-CD28-induced lymphocyte proliferation could be effectively blocked in 10% rat whole blood. In mice, 11 dose-dependently inhibited Staphylococcus aureus enterotoxin B-triggered IL-2 serum levels after oral dosing.

The structure of Ca2+ sensor Case16 reveals the mechanism of reaction to low Ca2+ concentrations.

Here we report the first crystal structure of a high-contrast genetically encoded circularly permuted green fluorescent protein (cpGFP)-based Ca(2+) sensor, Case16, in the presence of a low Ca(2+) concentration. The structure reveals the positioning of the chromophore within Case16 at the first stage of the Ca(2+)-dependent response when only two out of four Ca(2+)-binding pockets of calmodulin (CaM) are occupied with Ca(2+) ions. In such a "half Ca(2+)-bound state", Case16 is characterized by an incomplete interaction between its CaM-/M13-domains. We also report the crystal structure of the related Ca(2+) sensor Case12 at saturating Ca(2+) concentration. Based on this structure, we postulate that cpGFP-based Ca(2+) sensors can form non-functional homodimers where the CaM-domain of one sensor molecule binds symmetrically to the M13-peptide of the partner sensor molecule. Case12 and Case16 behavior upon addition of high concentrations of free CaM or M13-peptide reveals that the latter effectively blocks the fluorescent response of the sensor. We speculate that the demonstrated intermolecular interaction with endogenous substrates and homodimerization can impede proper functioning of this type of Ca(2+) sensors in living cells.

Discovery of 3-(1H-indol-3-yl)-4-[2-(4-methylpiperazin-1-yl)quinazolin-4-yl]pyrrole-2,5-dione (AEB071), a potent and selective inhibitor of protein kinase C isotypes.

A series of novel maleimide-based inhibitors of protein kinase C (PKC) were designed, synthesized, and evaluated. AEB071 (1) was found to be a potent, selective inhibitor of classical and novel PKC isotypes. 1 is a highly efficient immunomodulator, acting via inhibition of early T cell activation. The binding mode of maleimides to PKCs, proposed by molecular modeling, was confirmed by X-ray analysis of 1 bound in the active site of PKCalpha.

Advances in the structural biology, design and clinical development of VEGF-R kinase inhibitors for the treatment of angiogenesis.

Initial studies with angiogenesis inhibitors showed little clinical benefit. However, recently reported clinical studies in colorectal cancer have shown that bevacizumab, a vascular endothelial growth factor (VEGF) monoclonal antibody, in combination with cytotoxic therapy has positive effects on patient survival. Furthermore, the VEGF receptor kinase (VEGF-R) tyrosine kinase inhibitor, vatalanib, has also shown encouraging results in colorectal cancer, with molecular resonance imaging providing evidence that the anti-tumor efficacy was indeed the result of anti-angiogenic activity. Both of these agents are progressing in phase III trials. This proof of concept has stimulated the desire for second-generation VEGF-R inhibitors having an improved profile. Structural biology insight regarding the binding mode of protein kinase inhibitors is valuable for the design of molecules possessing superior selectivity, efficacy and tolerability. Towards this goal, we have developed a new series of VEGF-R2 kinase inhibitors, based upon an anthranilic acid amide scaffold. An X-ray crystal structure of a representative compound, AAL993 (ZK260253), in complex with the catalytic domain of diphosphorylated VEGF-R2 has revealed that this molecule binds to an inactive conformation of the protein. This binding mode, similar to that observed for the anti-leukemia drug, imatinib in complex with c-Abl kinase, may be responsible for the high selectivity of AAL993 and provides valuable insight for the design of further compounds.

Structure of rabbit-muscle glyceraldehyde-3-phosphate dehydrogenase.

The crystal structure of the tetrameric form of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isolated from rabbit muscle was solved at 2.4 A resolution after careful dynamic light-scattering experiments to find a suitable buffer for crystallization trials. The refined model has a crystallographic R factor of 20.3%. Here, the first detailed model of a mammalian GAPDH is presented. The cofactor NAD(+) (nicotinamide adenine dinucleotide) is bound to two subunits of the tetrameric enzyme, which is consistent with the negative cooperativity of NAD(+) binding to this enzyme. The structure of rabbit-muscle GAPDH is of interest because it shares 91% sequence identity with the human enzyme; human GAPDH is a potential target for the development of anti-apoptotic drugs. In addition, differences in the cofactor-binding pocket compared with the homology-model structure of GAPDH from the malaria parasite Plasmodium falciparum could be exploited in order to develop novel selective and potential antimalaria drugs.

Estrogen receptor modulators: identification and structure-activity relationships of potent ERalpha-selective tetrahydroisoquinoline ligands.

As part of a program aimed at the development of selective estrogen receptor modulators (SERMs), tetrahydroisoquinoline derivative 27 was discovered by high throughput screening. Successive replacements of the p-F substituent of 27 by an aminoethoxy side chain and of the 1-H of the tetrahydroisoquinoline core by a 1-Me group provided analogues 19 and 20. These compounds showed potencies in a cell-based reporter gene assay (ERE assay) varying between 0.6 and 20 nM and displayed antagonist behaviors in the MCF-7 human breast adenocarcinoma cell line with IC(50)s in the range of 2-36 nM. The effect of N-phenyl substituents on the activity and pharmacokinetic properties of tetrahydroisoquinoline analogues was explored. As a result of this investigation, two potent derivatives bearing a p-F N-aryl group, 19c and 20c, were discovered as candidates suitable for further profiling. To gain insight into the ligand-receptor interaction, the X-ray crystallographic structure of the 1-H tetrahydroisoquinoline derivative (R)-18a in complex with ERalpha-ligand binding domain (LBD)(301)(-)(553)/C-->S triple mutant was solved to 2.28 A. An overlay of this X-ray crystal structure with that reported for the complex of ERalpha-LBD(301)(-)(553)/carboxymethylated C and raloxifene (5) shows that both compounds bind to the same cleft of the receptor and display comparable binding modes, with differences being observed in the conformation of their "D-ring" phenyl groups.