Characterization of compound 584, an Abl kinase inhibitor with lasting effects

Background: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosomepositive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). Design and Methods: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotransplanted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. Results: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC50: 8 nM) and in cell-based assays (IC50: 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC50: 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. Conclusions: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former. ©2008 Ferrata Storti Foundation

Type II inhibitors targeting CDK2

Kinases can switch between active and inactive conformations of the ATP/Mg2+ binding motif DFG motif which has been explored by the development of type I or type II inhibitors. However, factors modulating DFG conformational remain poorly understood. We chose CDK2 as a model system to study the DFG out transition on a target that was thought to have an inaccessible DFG-out conformation using site directed mutagenesis of key residues identified in structural comparisons in conjunction with biochemical and biophysical characterization of the generated mutants. We identified key residues that facilitate the DFG out movement facilitating binding of type II inhibitors. However, surprisingly we also found that wild type CDK2 is able to bind type II inhibitors. Using protein crystallography structural analysis of the CDK2 complex with a aminopyrimidine-phenyl urea inhibitor (K03861) revealed a canonical type II binding mode and the first available type II CDK2 co-crystal structure. We found that the identified type II inhibitors compete with binding of activating cyclins. In addition, analysis of the binding kinetics of the identified inhibitors revealed slow dissociation off-rates. The study highlights the importance of residues that may be distant to the ATP binding pocket in modulating the energetics of the DFG out transition and hence inhibitor binding. The presented data provide also the foundation for a new class of slow off-rate cyclin competitive CDK2 inhibitors targeting the inactive DFG-out state of this important kinase target

Selective inhibitors of the Janus Kinase Jak3 – are they effective?

Jak3, together with Jak1, is involved in signal transduction initiated by several cytokines important in immune homeostasis and immune pathologies. Based on genetic evidence Jak3 has been considered to be an attractive target for immunosuppression. The approved Jak inhibitor tofacitinib (CP-690,550), originally introduced as a selective Jak3 inhibitor, also inhibited Jak1 and Jak2. The search for new selective Jak3 inhibitors has yielded several compounds whose profiles will be reviewed here. Implications on Jak3 as a therapeutic target are also discussed

A Fluorescence Lifetime-Based Assay for Abelson Kinase

We present a novel homogenous in vitro assay format, and apply it to the quantitative determination of the enzymatic activity of a tyrosine kinase. A single probe attached to a peptidic substrate responds with changes in its fluorescence lifetime, depending on whether or not a nearby tyrosine is phosphorylated. We utilize this effect to directly follow the enzymatic phosphorylation of the substrate, without having to resort to additional assay components such as antibodies. As an example for the application of this assay principle, we present results from the development of an assay for Abelson kinase (c-Abl) used for compound profiling. Adjustments in the peptide sequence would make this assay suitable to a wide variety of other tyrosine kinases

The Substrate-Activity-Screening methodology applied to receptor tyrosine kinases: A proof-of-concept study.

Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. A wide variety of disorders can arise as a consequence of abnormal kinase-mediated phosphorylation and numerous kinase inhibitors have earned their place as key components of the modern pharmacopeia. Although “traditional” kinase inhibitors typically act by preventing the interaction between the kinase and ATP, thus stopping substrate phosphorylation, an alternative approach consists in disrupting the protein-protein interaction between the kinase and its downstream partners. We herein report a proof-of-concept application of the Substrate Activity Screening methodology to Insulin-like Growth Factor 1 Receptor as a tool for the discovery of substrate-site-directed tyrosine kinase inhibitors

Promiscuity analysis of a kinase panel screen with designated p38 alpha inhibitors

Protein phosphorylation by kinases is of critical importance for the regulation of many cellular functions. When kinases are deregulated numerous biological processes are affected, which may cause a variety of diseases. Therefore, kinase inhibition plays an important role for therapeutic intervention. A number of kinase inhibitors have been approved as drugs, initially in oncology where promiscuous (multi-kinase) inhibitors were most efficacious. Exploring kinase inhibitor selectivity and promiscuity for therapy is among the most challenging aspects of kinase drug discovery. Herein, we thoroughly analyze a kinase profiling experiment in which 637 designated inhibitors of p38α MAP kinase (p38α) were tested against a panel of 60 kinases distributed across the human kinome. In this experiment, only 19% of the inhibitors were found to be promiscuous when the median p38α inhibition level was applied as an activity threshold. Promiscuous inhibitors had a median value of two targets per compound, and many of these inhibitors were only active against the p38α and closely related JNK3 enzymes. Promiscuity cliffs were identified and analyzed in a network representation revealing structural modifications that were implicated in triggering compound promiscuity. Taken together, the findings revealed a high degree of selectivity of designated p38α directed inhibitors although they target the ATP binding site that is largely conserved across the human kinome

Substrate profiling of IGF-1R and InsR: Identification of a potent pentamer substrate.

Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. As part of a study on the substrate requirements of Insulin-like Growth Factor 1 Receptor (IGF-1R) and Insulin Receptor (InsR), we evaluated and applied a universal assay system able to monitor the phosphorylation of unlabelled peptides of any length in real time. In contrast to already reported profiling methodologies, we were able to assess the kcat/KM ratio of peptides as short as tetramers. Notably, we were able to identify an efficient pentamer substrate that exhibited kinetic properties close to those of a 250-amino acid protein derived from IRS-1, a natural substrate of IGF-1R and InsR

Affinity classification of kinase inhibitors by mass spectrometric methods and validation using standard IC(50) measurements.

Protein kinases have emerged as a major drug target in the last years. Since more than 500 kinases are encoded in the human genome, cross-reactivity of a majority of kinase inhibitors causes problems. Tools are required for a rapid classification of inhibitors according to their affinity for a certain target to refine the search for new, more specific lead compounds. Mass spectrometry (MS) is increasingly used in pharmaceutical research and drug discovery to investigate protein-ligand interactions and determination of binding affinities. We present a comparison of different existing nanoelectrospray-MS based methods to quantify binding affinities and qualitatively rank, by competitive experiments, the affinity of several clinical inhibitors. We also present a new competitive method which is derived from our previous work for quantitative assessment of binding strengths (Wortmann et al., J. Mass Spectrom. 2008, 43(5), 600-608). The human kinases studied for this purpose were p38alpha (MAPK14) and LCK (lymphocyte specific kinase), and their interaction with 17 known small molecule kinase inhibitors was probed. Moreover, we present a new method to differentiate type I from type II inhibitors (Liu, Y.; Gray, N. S. Nat. Chem. Biol. 2006, 2(7), 358-364) based on a kinetic experiment with direct MS read-out of the noncovalent complex between the human kinase and the inhibitor. This method was successfully applied to p38alpha binding to BIRB796, as well as to a BIRB796 analogue. Quantitative determination of the binding strength is also described. The results of our competitive experiments for the affinity classification of different inhibitors, as well as the results for the kinetic study, are in good agreement with IC(50) measurements and data found in the literature

Savanna fires and their impact on net ecosystem productivity in North Australia

Savannas comprise a large area of the global land surface and are subject to frequent disturbance through fire. The role of fire as one of the primary natural carbon cycling mechanisms is a key issue in considering global change feedbacks. The savannas of Northern Australia burn regularly and we aimed to determine their annual net ecosystem productivity (NEP) and the impact of fire on productivity. We established a long-term eddy covariance flux tower at Howard Springs, Australia and present here 5 years of data from 2001 to 2005. Fire has direct impacts through emissions but also has indirect effects through the loss of productivity due to reduced functional leaf area index and the carbon costs of rebuilding the canopy. The impact of fire on the canopy latent energy exchange was evident for 40 days while the canopy was rebuilt; however, the carbon balance took approximately 70 days to recover. The annual fire free NEP at Howard Springs was estimated at −4.3 t C ha−1 yr−1 with a range of −3.5 to −5.1 t C ha−1 yr−1 across years. We calculated the average annual indirect fire effect as +0.7 t C ha−1 yr−1 using a neural network model approach and estimated average emissions of fine and coarse fuels as +1.6 t C ha−1 yr-1. This allowed us to calculate a net biome production of −2.0 t C ha−1 yr-1. We then partitioned this remaining sink and suggest that most of this can be accounted for by woody increment (1.2 t C ha−1 yr-1) and shrub encroachment (0.5 t C ha−1 yr-1). Given the consistent sink at this site, even under an almost annual fire regime, there may be management options to increase carbon sequestration by reducing fire frequency