Thomas Machleidt

Identification of a new JNK inhibitor targeting the JNK-JIP interaction site

JNK is a stress-activated protein kinase that modulates pathways implicated in a variety of disease states. JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases. A minimal peptide region derived from JIP1 is able to inhibit JNK activity both in vitro and in cell. We report here a series of small molecules JIP1 mimics that function as substrate competitive inhibitors of JNK. One such compound, BI-78D3, dose-dependently inhibits the phosphorylation of JNK substrates both in vitro and in cell. In animal studies, BI-78D3 not only blocks JNK dependent Con A-induced liver damage but also restores insulin sensitivity in mouse models of type 2 diabetes. Our findings open the way for the development of protein kinase inhibitors targeting substrate specific docking sites, rather than the highly conserved ATP binding sites. In view of its favorable inhibition profile, selectivity, and ability to function in the cellular milieu and in vivo, BI-78D3 represents not only a JNK inhibitor, but also a promising stepping stone toward the development of an innovative class of therapeutics.

Design, Synthesis, and Structure-Activity Relationship of Substrate Competitive, Selective, and in Vivo Active Triazole and Thiadiazole inhibitors of the c-Jun N-Terminal Kinase

We report comprehensive structure-activity relationship studies on a novel series of c-Jun N-terminal kinase (JNK) inhibitors. The compounds are substrate competitive inhibitors that bind to the docking site of the kinase. The reported medicinal chemistry and structure-based optimizations studies resulted in the discovery of selective and potent thiadiazole JNK inhibitors that display promising in vivo activity in mouse models of insulin insensitivity.

Protein labeling with FlAsH and ReAsH.

The ability to image biochemical and phenotypical changes in living cells has become crucial for the investigation and understanding of the molecular mechanisms that govern all physiological cellular functions in health and disease. Genetically encoded reporters derived from fluorescent proteins (FPs) have proved to be extremely useful for localization and interaction studies in living cells. However, the large size and spectral properties of FP impose certain limitations for their use. The recently developed Fluorescein Arsenical Hairpin (FlAsH/tetracysteine) binder technology emerged as a promising alternative to FP for protein labeling and cellular localization studies. The combination of a small genetically encoded peptide tag with a small molecule detection reagent makes this technology particularly suitable for the investigation of biochemical changes in living cells that are difficult to approach with fluorescent proteins as molecular tags. We describe the practical application of this technology to image protein dynamics in living cells.

Synthesis and optimization of thiadiazole derivatives as a novel class of substrate competitive c-Jun N-terminal kinase inhibitors.

A series of thiadiazole derivatives has been designed as potential allosteric, substrate competitive inhibitors of the protein kinase JNK. We report on the synthesis, characterization and evaluation of a series of compounds that resulted in the identification of potent and selective JNK inhibitors targeting its JIP-1 docking site.

Development of LanthaScreen™ Cellular Assays for Key Components within the PI3K/AKT/mTOR Pathway

The PI3K/AKT/mTOR pathway is central to cell growth and survival, cell cycle regulation, and programmed cell death. Aberrant activation of this signaling cascade is linked to several disease states, and thus many components of the pathway are attractive targets for therapeutic intervention. However, the considerable degree of complexity, crosstalk, and feedback regulation that exists within the pathway (especially with respect to the regulation of mTOR and its complexes) underscores the need for a comprehensive set of cell-based assays to properly identify and characterize small-molecule modulators. Here, the development and application of time-resolved Förster resonance energy transfer (TR-FRET)—based assays to enable the phosphoprotein analysis of key pathway components in a cellular format are reported. The LanthaScreen™ cellular assay platform uses FRET between a terbium-labeled phosphorylation site-specific antibody and an expressed green fluorescent protein fusion of particular kinase substrate and provides an assay readout that is ratiometric, robust, and amenable to high-throughput screening applications. Assays specific for 5 different targets within the pathway are highlighted: Ser183 and Thr246 on the proline-rich AKT substrate 40 kDa (PRAS40), Ser457 on programmed cell death protein 4 (PDCD4), and Thr308 and Ser473 on AKT. Each assay was evaluated under various experimental conditions and individually optimized for performance. Known pathway agonists and a small panel of commercially available compounds were also used to complete the assay validation. Taken together, these data demonstrate the utility of a related set of cell-based assays to interrogate PI3K/AKT/mTOR signaling and provide a template for the development of similar assays for other targets. (Journal of Biomolecular Screening 2009:121-132)

Design and characterization of a potent and selective dual ATP- and substrate-competitive subnanomolar bidentate c-Jun N-terminal kinase (JNK) inhibitor.

c-Jun N-terminal kinases (JNKs) represent valuable targets in the development of new therapies. Present on the surface of JNK is a binding pocket for substrates and the scaffolding protein JIP1 in close proximity to the ATP binding pocket. We propose that bidentate compounds linking the binding energies of weakly interacting ATP and substrate mimetics could result in potent and selective JNK inhibitors. We describe here a bidentate molecule, 19, designed against JNK. 19 inhibits JNK kinase activity (IC(50) = 18 nM; K(i) = 1.5 nM) and JNK/substrate association in a displacement assay (IC(50) = 46 nM; K(i) = 2 nM). Our data demonstrate that 19 targets for the ATP and substrate-binding sites on JNK concurrently. Finally, compound 19 successfully inhibits JNK in a variety of cell-based experiments, as well as in vivo where it is shown to protect against Jo-2 induced liver damage and improve glucose tolerance in diabetic mice.

Discovery of 2-(5-nitrothiazol-2-ylthio)benzo[d]thiazoles as novel c-Jun N-terminal kinase inhibitors.

A new series of 2-thioether-benzothiazoles has been synthesized and evaluated for JNK inhibition. The SAR studies led to the discovery of potent, allosteric JNK inhibitors with selectivity against p38.

Design, synthesis, and structure-activity relationship studies of thiophene-3-carboxamide derivatives as dual inhibitors of the c-Jun N-terminal kinase.

We report comprehensive structure-activity relationship studies on a novel series of c-Jun N-terminal kinase (JNK) inhibitors. Intriguingly, the compounds have a dual inhibitory activity by functioning as both ATP and JIP mimetics, possibly by binding to both the ATP binding site and to the docking site of the kinase. Several of such novel compounds display potent JNK inhibitory profiles both in vitro and in cell.

Multiplexing of Pathway-Specific β-Lactamase Reporter Gene Assays by Optical Coding With Qtracker® Nanocrystals

Reporter assays are widely used in research and drug discovery for analysis of signaling pathways in a cell-based format. Traditionally, reporter gene assays are run in a single-parameter mode, interrogating only 1 pathway per sample. To enable more complex assay formats for pathway analysis, the authors developed a multiplexed reporter cell-based assay that combines optical encoding with a β-lactamase reporter gene readout. The optical encoding is achieved by peptide-mediated delivery of quantum dots into reporter cell lines. Using different quantum dots, the authors were able to simultaneously analyze multiple signaling pathways in the same sample using fluorescence microscopy or flow cytometry. They selected 3 β-lactamase reporter cell lines for the analysis of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interferon gamma (IFN-γ) induced signaling to perform proof-of-principle experiments. The analysis demonstrates that this multiplexed assay allows the reliable detection of ligand-specific activation patterns as well as pathway-specific inhibitors. This method provides a template for the development of novel assay designs that enable the analysis of complex signaling networks involving multiple signaling pathways as well as cell-specific pathways in heterotypic cell models. (Journal of Biomolecular Screening 2009:845-852)