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NOD2 (nucleotide-binding oligomerization domain-containing protein 2) is an internal pattern recognition receptor that recognizes bacterial peptidoglycan and stimulates host immune responses. Dysfunction of NOD2 pathway has been associated with a number of autoinflammatory disorders. To date, direct inhibitors of NOD2 have not been described due to technical challenges of targeting the oligomeric protein complex. Receptor interacting protein kinase 2 (RIPK2) is an intracellular serine/threonine/tyrosine kinase, a key signaling partner, and an obligate kinase for NOD2. As such, RIPK2 represents an attractive target to probe the pathological roles of NOD2 pathway. To search for selective RIPK2 inhibitors, we employed virtual library screening (VLS) and structure based design that eventually led to a potent and selective RIPK2 inhibitor 8 with excellent oral bioavailability, which was used to evaluate the effects of inhibition of RIPK2 in various in vitro assays and ex vivo and in vivo pharmacodynamic models.

A series of 4-amino-6-benzimidazole-pyrimidines was designed to target lymphocyte-specific tyrosine kinase (Lck), a member of the Src-family kinases (SFKs). These type II inhibitors were optimized using a cellular Lck-dependent proliferation assay and are capable of inhibiting Lck at single-digit nanomolar concentrations. This scaffold is likely to serve a valuable template for developing potent inhibitors of a number of SFKs.

The discovery, synthesis, and optimization of compound 1 from a high-throughput screening hit to highly potent and selective peroxisome proliferator-activated receptor delta (PPARdelta) agonists are reported. The synthesis and structure-activity relationship in this series are described in detail. On the basis of a general schematic PPAR pharmacophore model, scaffold 1 was divided into headgroup, linker, and tailgroup and successively optimized for PPAR activation using in vitro PPAR transactivation assays. A (2-methylphenoxy)acetic acid headgroup, a flexible linker, and a five-membered heteroaromatic center ring with two hydrophobic aryl substituents were required for efficient and selective PPARdelta activation. The fine-tuning of these aryl substituents led to an array of highly potent and selective compounds such as compound 38c, displaying an excellent pharmacokinetic profile in mouse. In an in vivo acute dosing model, selected members of this array were shown to induce the expression of pyruvate dehydrogenase kinase-4 (PDK4) and uncoupling protein-3 (UCP3), genes that are known to be involved in energy homeostasis and regulated by PPARdelta in skeletal muscle.

A series of 4-amino-6-benzimidazole-pyrimidines was designed to target lymphocyte-specific tyrosine kinase (Lck), a member of the Src kinase family. Highly efficient parallel syntheses were devised to prepare analogues for SAR studies. A number of these 4-amino-6-benzimidazole-pyrimidines exhibited single-digit nanomolar IC50s against Lck in biochemical and cellular assays. These 4-amino-6-benzimidazole-pyrimidines represent a new class of tyrosine kinase inhibitors.

A novel series of 2-amino-6-carboxamidobenzothiazole was discovered to have potent Lck inhibitory properties. A highly efficient chemistry was developed. Also described are the detailed SAR study and the BaF3 cell line profiling for this series.

A series of PPARdelta-selective agonists was investigated and optimized for a favorable in vivo pharmacokinetic profile. Isoxazole LCI765 (17d) was found to be a potent and selective PPARdelta agonist with good in vivo PK properties in mouse (C(max)=5.1 microM, t(1/2)=3.1 h). LCI765 regulated expression of genes involved in energy homeostasis in relevant tissues when dosed orally in C57BL6 mice. A co-crystal structure of compound LCI765 and the LBD of PPARdelta is discussed.

The interaction of the zinc finger protein WT1 with RNA aptamers has been investigated using a quantitative binding assay, and the results have been compared to those from a previous study of the DNA binding properties of this protein. A recombinant peptide containing the four zinc fingers of WT1 (WT1-ZFP) binds to representatives of three specific families of RNA aptamers with apparent dissociation constants ranging from 13.8 +/- 1.1 to 87.4 +/- 10.4 nM, somewhat higher than the dissociation constant of 4.12 +/- 0.4 nM for binding to DNA. An isoform that contains an insertion of three amino acids between the third and fourth zinc fingers (WT1[+KTS]-ZFP) also binds to these RNAs with slightly reduced affinity (the apparent dissociation constants ranging from 22.8 to 69.8 nM) but does not bind to DNA. The equilibrium binding of WT1-ZFP to the highest-affinity RNA molecule was compared to the equilibrium binding to a consensus DNA molecule as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1-ZFP with both nucleic acids is an entropy-driven process. Binding of WT1-ZFP to RNA has a pH optimum that is narrower than that observed for binding to DNA. Binding of WT1-ZFP to DNA is optimal at 5 mM MgCl(2), while the highest affinity for RNA was observed in the absence of MgCl(2). Binding of WT1 to both nucleic acid ligands is sensitive to increasing monovalent salt concentration, with a greater effect observed for DNA than for RNA. Point mutations in the zinc fingers associated with Denys-Drash syndrome have dramatically different effects on the interaction of WT1-ZFP with DNA, but a consistent and modest effect on the interaction with RNA. The role of RNA sequence and secondary structure in the binding of WT1-ZFP was probed by site-directed mutagenesis. Results indicate that a hairpin loop is a critical structural feature required for protein binding, and that some consensus nucleotides can be substituted provided proper base pairing of the stem of the hairpin loop is maintained.

We have characterized the sperm nuclear basic proteins (SNBPs) of the sticklebacks in the suborder Gasterosteoidei. The complete amino acid sequence of the protamines from Aulorhynchus flavidus, Pungitius pungitius, Gasterosteus aculeatus, (anadromous) and G. wheatlandi, as well as the sequences of the protamines of several species pairs of freshwater G. aculeatus, have been determined. Analysis of the primary structure of these proteins has shown that: a) despite the relatively low amino acid complexity and small molecular mass of these basic proteins, they are very good molecular markers at the generic level. The bootstrap parsimony analysis using their sequences provides a phylogenetic relationship for the old anadromous species of Gasterosteoidei which is identical to that obtained from morphological and behavioral analysis; b) the comparison of the sequences also suggests that protamines from the suborder Gasterosteoidei have most likely evolved from a common gene in the early Acanthopterygii by an extension of the carboxy terminal portion of the molecule; c) protamines are not good markers for recent postglacial freshwater isolates of G. aculeatus. However, in the unique case of Enos Lake (British Columbia), we have been able to detect an additional minor protamine component in the benthic forms of G. aculeatus that is not present in the limnetic forms. Thus, this new protamine must have appeared during the past 12,000 years concomitantly with the speciation of benthics and limnetics in this lake.

We have reconstituted nucleosome core particles from reversed-phase-HPLC-purified chicken erythrocyte core histones and 145 bp random-sequence DNA fragments. Characterization of the resulting nucleoprotein complexes by sedimentation velocity, CD and DNase I footprinting showed that they are structurally indistinguishable from native nucleosome core particles. Furthermore, we have shown that the ability to reproduce these native-like structural features in these reconstituted nucleosome core particles is basically independent of the biological source or the method used (i.e. salt versus acid) for the extraction of histones before their HPLC fractionation. The usefulness and relevance of this approach for the reconstitution of native-like chromatin structures from histone types (histone variants/post-translationally modified histones), which are usually available only in relatively small amounts, is discussed.

A series of highly potent and selective PPARdelta agonists is described using the known non-selective ligand GW2433 as a structural template. Compound 1 is bioavailable, potent (10 nM), and shows no cross-activity with other PPAR subtypes up to 10 microM, making it a useful tool in studying the biological effects of selective PPARdelta activation.

We report the identification of a novel series of trisubstituted isoxazoles as PPAR activators from a high-throughput screen. A series of structural optimizations led to improved efficacy and excellent functional receptor selectivity for PPARdelta. The isoxazoles represent a series of agonists which display a scaffold that lies outside the typical PPAR agonist motif.

Using equilibrium dialysis and sedimentation velocity analysis, we have characterized the binding of the anti-tumor drug daunomycin to chicken erythrocyte chromatin before and after depletion of linker histones and to its constitutive DNA under several ionic strengths (5, 25, and 75 mm NaCl). The equilibrium dialysis experiments reveal that the drug binds cooperatively to both the chromatin fractions and to the DNA counterpart within the range of ionic strength used in this study. A significant decrease in the binding affinity was observed at 75 mm NaCl. At any given salt concentration, daunomycin exhibits higher binding affinity for DNA than for linker histone-depleted chromatin or chromatin (in decreasing order). Binding of daunomycin to DNA does not significantly affect the sedimentation coefficient of the molecule. This is in contrast to binding to chromatin and to its linker histone-depleted counterpart. In these instances, preferential binding of the drug to the linker DNA regions induces an unfolding of the chromatin fiber that is followed by aggregation, presumably because of histone-DNA interfiber interactions.

International audience; In Podospora anserina a linear plasmid with telomeric ends behaves as an artificial acentric minichromosome. Transformation is at least 100 times more efficient than with integrative vectors. Genomic DNA was inserted in this plasmid in vitro and the mixture used to transform a leu1-1 strain. Many fungal clones containing the leu1 gene as a genomic insert in the linear plasmid were identified. The leu1 gene was rescued as a circular plasmid in Escherichia coli demonstrating that a direct cloning procedure can be applied for the fungus P. anserina. The conservation of telomeric sequences among filamentous fungi suggests that a telomere-based linear plasmid could provide a general cloning vector for filamentous fungi.

We have reconstituted oligonucleosome complexes containing histone H1 starting from a synthetic DNA template, consisting of 12 tandemly arranged 208-base pair fragments of the 5 S rRNA gene, purified HeLa histone octamers, and histone H1. A ratio of histone H1 per histone octamer used in the reconstitution (0.8–0.9 mol of histone H1/mol of histone octamer) similar to that observed in vivo was used. The reconstituted chromatin complexes exhibit a salt-dependent folding, which is almost indistinguishable from that exhibited by chromatin fragments obtained from nuclease digestion of native chromatin. The folding of this reconstituted chromatin complex seems to be rather independent of the symmetrical or asymmetrical position occupied by H1 in the individual nucleosomes. Binding of histone H1 to the oligonucleosome complexes, under the stoichiometric binding conditions used, had no inhibitory effect on the transcriptional potential of these complexes.

Vegetative incompatibility in fungi results from the control of heterokaryon formation by the genes present at het loci. Coexpression of antagonistic het genes in the same hyphae leads to a lethal process. In Podospora anserina, self-incompatible strains containing nonallelic incompatible genes in the same nucleus are inviable as the result of a growth arrest and a lytic process. Mutations in suppressor genes (mod genes) can restore the viability. These mod mutations also interfere with developmental processes, which suggests common steps between the incompatibility reaction and cellular differentiation. The mod-A locus, responsible for growth arrest in the self-incompatible strains, is also involved in the control of the development of female organs. The mod-A gene was isolated. An open reading frame 687 amino acids long was identified. The MOD-A-encoded polypeptide is rich in proline residues, which are clustered in a domain containing a motif that displays similarity to SH3-binding motifs, which are known to be involved in protein-protein interactions. Construction of a strain deleted for mod-A confirmed that the product of this gene involved in differentiation is a key regulator of growth arrest associated with vegetative incompatibility.