Arabidopsis transportin1 is the nuclear import receptor for the circadian clock-regulated RNA-binding protein AtGRP7

Ziemienowicz A, Haasen D, Staiger D, Merkle T. Arabidopsis transportin1 is the nuclear import receptor for the circadian clock-regulated RNA-binding protein AtGRP7. Plant Molecular Biology. 2003;53(1/2):201-212.We characterized the Arabidopsis orthologue of the human nuclear import receptor transportin1 (TRN1). Like the human receptor, Arabidopsis TRN1 recognizes nuclear import signals on proteins that are different from the classical basic nuclear localization signals. The M9 domain of human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the prototype of such signals. We show that At TRN1 binds to similar domains in hnRNP-like proteins from plants. At TRN1 also interacts with human hnRNP A1 and with yeast Nab2p, two classical import cargo proteins of transportin in these organisms. Like all nuclear transport receptors of the importin-Ã family, At TRN1 binds to the regulatory GTPase Ran from Arabidopsis . We demonstrated that the amino terminus of At TRN1 is necessary for this interaction. Recombinant At TRN1 conferred nuclear import of fluorescently labelled BSA-M9 peptide conjugates in permeabilized HeLa cells, functionally replacing human TRN1 in these in vitro nuclear import assays. We identified three plant substrate proteins that interact with At TRN1 and contain M9-like domains: a novel Arabidopsis hnRNP that shows high similarity to human hnRNP A1 and two small RNA-binding proteins from Arabidopsis , At GRP7 and At GRP8. Nuclear import activity of the M9-like domains of these plant proteins was demonstrated in vivo by their ability to confer partial nuclear re-localisation of a GFP fusion protein containing a nuclear export signal. In addition, fluorescently labelled At GRP7 was specifically imported into nuclei of permeabilized HeLa cells by Arabidopsis At TRN1 and human TRN1. These results suggest that the transportin-mediated nuclear import pathway is highly conserved between man, yeast and plants

How phenotypic screening influenced drug discovery – lessons from 5 years practice

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High Content Screening of CXCR2-Dependent Signalling Pathways

Stimulation of CXC-type chemokine receptor 2 (CXCR2)-transfected cells by Gro-? or IL-8 induced (i) CXCR2 internalization, (ii) phosphorylation of ERK1/2 (pERK) and (iii) translocation of nuclear factor of activated T cells (NFAT) into the nucleus. Employing high content screening (HCS; i.e. fluorimetric imaging combined with image analysis) these three ligand-induced events were quantified by using a CXCR2-specific antibody, an antibody recognizing phosphorylated ERK1/2 (pERK) and a red fluorescent protein (RFP) in fusion to transiently overexpressed NFAT, respectively. As an RFP, we applied a recently developed mutant of an Entacmaea quadricolor fluorescent protein with favorable properties for HCS, such as high fluorescence brightness, photostability, large Stokes shift, and stability with regard to formaldehyde.Receptor internalization was closely coupled with ERK signalling both when analyzed in regard of stimulation by physiological CXCR2 ligands and when observed in the presence of antagonistic test compounds. A means of increasing the throughput or of broadening the pharmacological characterization of test compounds is the use of multiplexed imaging. Indeed, CXCR2 internalization could be multiplexed with the NFAT nuclear translocation by fixation at ~45 min after Gro-? stimulation. This multiplexing demonstrated that Gro-?-induced CXCR2 internalization was tightly correlated with Gro-?-induced NFAT translocation, also on the single cell level.The analysis of ERK phosphorylation, NFAT translocation and receptor internalization enabled the profiling of antagonistic test compounds with respect to G-protein signalling and possible receptor desensitization liabilities

Identification and optimisation of a 4',5-bisthiazole series of selective phosphatidylinositol-3 kinase alpha inhibitors

Abstract: Exploring the affinity-pocket binding moiety of a 2-aminothiazole (S)-proline-amide-urea series of selective PI3Kα inhibitors using a parallel-synthesis approach led to the identification of a novel 4',5-bisthiazole sub-series. The synthesis and optimisation of both the affinity pocket and (S)-proline amide moieties within this 4',5-bisthiazole sub-series are described. From this work a number of analogues, including 14 (A66) and 24, were identified as potent and selective PI3Kα inhibitor in vitro tool compounds

A Drug Resistance Screen Using a Selective MET Inhibitor Reveals a Spectrum of Mutations That Partially Overlap with Activating Mutations Found in Cancer Patients

The emergence of drug resistance is a primary concern in any cancer treatment, including with targeted kinase inhibitors as exemplified by the appearance of Bcr-Abl point mutations in chronic myeloid leukemia (CML) patients treated with imatinib. In vitro approaches to identify resistance mutations in Bcr-Abl have yielded mutation spectra that faithfully recapitulated clinical observations. To predict resistance mutations in the receptor tyrosine kinaseMETthat could emerge during inhibitor treatment in patients, we conducted a resistance screen in BaF3 TPR-METcells using the novel selectiveMETinhibitor NVP-BVU972. The observed spectrumof mutations in resistant cells was dominated by substitutions of tyrosine 1230 but also included other missense mutations and partially overlapped with activating MET mutations that were previously described in cancer patients. Cocrystallization of the MET kinase domain in complex with NVP-BVU972 revealed a key role for Y1230 in binding of NVP-BVU972, as previously reported for multiple other selective MET inhibitors. A second resistance screen in the same format with the MET inhibitor AMG 458 yielded a distinct spectrum of mutations rich in F1200 alterations, which is consistent with a different predicted binding mode. Our findings suggest that amino acid substitutions in the MET kinase domain of cancer patients need to be carefully monitored before and during treatment with MET inhibitors, as resistance may preexist or emerge. Compounds binding in the same manner as NVP-BVU972 might be particularly susceptible to the development of resistance through mutations in Y1230, a condition that may be addressed by MET inhibitors with alternative binding modes

Discovery of novel pyrrolidineoxy-substituted heteroaromatics as potent and selective PI3K delta inhibitors with improved physicochemical properties

In the recent years, PI3Kδ has emerged as a promising target for the treatment of B- and T-cell mediated inflammatory diseases. A detailed analysis of our previously reported 4,6-diaryl quinazoline PI3Kδ inhibitor series revealed that the activity in cellular assays was logP dependent, with an optimum logP range between 2-3. We discovered novel analogues in this lipophilicity space that feature a chiral pyrrolidineoxy-group instead of one of the aromatic rings. Compared to 4,6-diaryl quinazolines, these Fsp3 enriched derivatives retain potency and selectivity towards PI3Kδ, yet their permeability profile is improved and molecular weight as well as PSA are reduced. These modifications offer additional possibilities for derivative generation in a good physicochemical property space and thus increase the chances to identify a clinical candidate

A High-Throughput Screening Identifies MICU1 Targeting Compounds

Mitochondrial Ca2+ uptake depends on the mitochondrial calcium uniporter (MCU), a highly selective tetrameric channel of the inner mitochondrial membrane (IMM), composed of pore forming and of regulatory subunits. Here, we screened a library of 44k non-proprietary compounds for their ability to modulate mitochondrial Ca2+ uptake. Two of them decreased mitochondrial Ca2+ influx both in cell lines and in isolated mouse skeletal muscle fibers. A closer inspection revealed that these molecules directly bound a specific cleft in the MCU complex component and positive regulator MICU1. In MICU1-silenced or deleted cells, the inhibitory effect of the two compounds on Ca2+ influx was lost, demonstrating that MICU1 is required for compound activity. Moreover, in MICU1-KO cells overexpressing a mutant isoform of MICU1, in which critical amino acids of the predicted binding cleft were mutated, the two compounds were unable to decrease mitochondrial Ca2+ uptake. Finally, the compounds were tested ex vivo unravelling a role for mitochondrial Ca2+ uptake in muscle growth. These compounds represent leading molecules for the development of MICU1-targeting drugs

Targeting Plasmodium phosphatidylinositol-4 kinase for the treatment, prevention and elimination of malaria

Achieving the goal of malaria elimination is vitally dependent on identifying validated drug targets that are active across all stages of the Plasmodium lifecycle. Here, we identify phosphatidylinositol-4 kinase (PfPI4K) as the target of the imidazopyrazines, a novel antimalarial compound class that potently inhibits the intracellular development of multiple Plasmodium species at each stage of infection of the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in several rodent malaria models. These compounds are also active against blood-stage field isolates of the major human malaria pathogens, P. falciparum and P. vivax, and inhibit liver stage hypnozoites in the human and simian parasite P. cynomolgi. Evolved resistance, full genome-scanning and genome editing experiments in intra-erythrocytic stages as well as biochemical data, show that imidazopyrazines exert their potent antimalarial activity through interaction with the ATP-binding pocket of the lipid kinase. Inhibition of PfPI4K, alters the intracellular distribution of phosphatidylinositol-4 phosphate, the PI4K product, and interferes with cytokinesis via a Rab11A-dependent pathway. Collectively, our data define PfPI4K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify antimalarial drugs with an ideal pharmacological profile for the prevention, treatment and elimination of malaria

Targeting Plasmodium PI(4)K to eliminate malaria.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria