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[Image: see text] The HIV-1 nucleocapsid (NC) protein is a small basic DNA and RNA binding protein that is absolutely necessary for viral replication and thus represents a target of great interest to develop new anti-HIV agents. Moreover, the highly conserved sequence offers the opportunity to escape the drug resistance (DR) that emerged following the highly active antiretroviral therapy (HAART) treatment. On the basis of our previous research, nordihydroguaiaretic acid 1 acts as a NC inhibitor showing moderate antiviral activity and suboptimal drug-like properties due to the presence of the catechol moieties. A bioisosteric catechol replacement approach led us to identify the 5-dihydroxypyrimidine-6-carboxamide substructure as a privileged scaffold of a new class of HIV-1 NC inhibitors. Hit validation efforts led to the identification of optimized analogs, as represented by compound 28, showing improved NC inhibition and antiviral activity as well as good ADME and PK properties.

Ciliary neurotrophic factor (CNTF) is a neurotrophic cytokine able to induce appetite reduction, weight loss and antidiabetic effects. However, its susceptibility to neutralizing anti-CNTF antibodies in patients hampered its use for treatment of human obesity and diabetes. In addition, CNTF has a very short plasma half-life, which limits its use as a therapeutic agent. Solutions, directed to prolong its in vivo effects, vary from the implantation of encapsulated secreting cells to identification of more active variants or chemical modification of the protein itself. PEGylation is a widely used modification for shielding proteins from circulating antibodies and for increasing their plasma half-life. Here, we have selected DH-CNTF, a CNTF variant which has a 40-fold higher affinity for the CNTF receptor α accompanied by an increased activity in cellular assays. The PEGylated DH-CNTF retained the biological activity of native protein in vitro and showed a significant improvement of pharmacokinetic parameters. In an acute model of glucose tolerance, the PEG-DH-CNTF was able to reduce the glycemia in diet-induced obese animals, with a performance equaled by a 10-fold higher dose of DH-CNTF. In addition, the PEGylated DH-CNTF analog demonstrated a more potent weight loss effect than the unmodified protein, opening to the use of CNTF as weight reducing agent with treatment regimens that can better meet patient compliance thanks to reduced dosing schedules.

[Image: see text] The application of class I HDAC inhibitors as cancer therapies is well established, but more recently their development for nononcological indications has increased. We report here on the generation of improved class I selective human HDAC inhibitors based on an ethylketone zinc binding group (ZBG) in place of the hydroxamic acid that features the majority of HDAC inhibitors. We also describe a novel set of HDAC3 isoform selective inhibitors that show stronger potency and selectivity than the most commonly used HDAC3 selective tool compound RGFP966. These compounds are again based on an alternative ZBG with respect to the ortho-anilide that is featured in HDAC3 selective compounds reported to date.

The blood-brain barrier (BBB) is responsible for the homeostasis between the cerebral vasculature and the brain and it has a key role in regulating the influx and efflux of substances, in healthy and diseased states. Stem cell technology offers the opportunity to use human brain-specific cells to establish in vitro BBB models. Here, we describe the establishment of a human BBB model in a two-dimensional monolayer culture, derived from human induced pluripotent stem cells (hiPSCs). This model was characterized by a transendothelial electrical resistance (TEER) higher than 2000 Ω∙cm2 and associated with negligible paracellular transport. The hiPSC-derived BBB model maintained the functionality of major endothelial transporter proteins and receptors. Some proprietary molecules from our central nervous system (CNS) programs were evaluated revealing comparable permeability in the human model and in the model from primary porcine brain endothelial cells (PBECs).

The identification of a new series of P. falciparum growth inhibitors is described. Starting from a series of known human class I HDAC inhibitors a SAR exploration based on growth inhibitory activity in parasite and human cells-based assays led to the identification of compounds with submicromolar inhibition of P. falciparum growth (EC50500 nM) and good selectivity over the activity of human HDAC in cells (up to50-fold). Inhibition of parasital HDACs as the mechanism of action of this new class of selective growth inhibitors is supported by hyperacetylation studies.

Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.

We describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis C virus (HCV) envelope 2 (E2) glycoprotein responses in mammals through electrical gene transfer (EGT) of plasmid DNA into muscle fibers. Intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (HVR1) peptide mimic fused at the N terminus of the E2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, induced more than 10-fold-higher expression levels in the transfected mouse tissue. As a result of this substantial increment of in vivo antigen production, the humoral response induced in mice, rats, and rabbits ranged from 10- to 30-fold higher than that induced by conventional naked DNA immunization. Consequently, immune sera from EGT-treated mice displayed a broader cross-reactivity against HVR1 variants from natural isolates than sera from injected animals that were not subjected to electrical stimulation. Cellular response against E2 epitopes specific for helper and cytotoxic T cells was significantly improved by EGT. The EGT-mediated enhancement of humoral and cellular immunity is antigen independent, since comparable increases in antibody response against ciliary neurotrophic factor or in specific anti-human immunodeficiency virus type 1 gag CD8+T cells were obtained in rats and mice. Thus, the method described potentially provides a safe, low-cost treatment that may be scaled up to humans and may hold the key for future development of prophylactic or therapeutic vaccines against HCV and other infectious diseases.

Leptin is an adipocyte-secreted hormone that regulates body weight and exerts effects on hematopoiesis, reproduction, and immunity. The leptin receptor (OBR) shares sequence similarity and signaling capabilities with receptors for cytokines of the ciliary neurotrophic factor (CNTF) family. Our previous finding that CNTF and leptin exert similar anti-obesity effects and activate common neuronal signaling pathways, prompted us to investigate whether leptin may share with CNTF the ability to regulate the expression of specific neuronal genes. To this end, we established a cell line, derived from the murine septal cholinergic neuronal cell line SN-56, which stably expresses OBR. In this cell line, termed SN-56/OBR, leptin induces STAT transcription factor activation and STAT-dependent reporter gene expression in a manner similar to that of CNTF. Furthermore, in SN-56/OBR cells both CNTF and leptin produce changes in neurotransmitter and neuropeptide phenotype characteristic of cholinergic neurons, such as an increase in choline acetyltransferase and vasoactive intestinal polypeptide, and a decrease in neuropeptide Y expression. SN-56/OBR cells thus constitute an interesting new model system to investigate leptin action in cells of central nervous system origin. Possible physiological implications of OBR's intrinsic ability to regulate cholinergic phenotypic markers are discussed.

Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.

Target-derived neurotrophic factors are assumed to regulate motoneuron cell death during development but remain unspecified. Motoneuron cell death in the spinal nucleus of the bulbocavernosus (SNB) of rats extends postnatally and is controlled by androgens. We exploited these features of the SNB system to identify endogenously produced trophic factors regulating motoneuron survival. Newborn female rat pups were treated with the androgen, testosterone propionate, or the oil vehicle alone. In addition, females received trophic factor antagonists delivered either into the perineum (the site of SNB target muscles) or systemically. Fusion molecules that bind and sequester the neurotrophins (trkA-IgG, trkB-IgG, and trkC-IgG) were used to block activation of neurotrophin receptors, and AADH-CNTF was used to antagonize signaling through the ciliary neurotrophic factor receptor-α (CNTFRα). An acute blockade of trkB, trkC, or CNTFRα prevented the androgenic sparing of SNB motoneurons when antagonists were delivered to the perineum. Trophic factor antagonists did not significantly reduce SNB motoneuron number when higher doses were injected systemically. These findings demonstrate a requirement for specific, endogenously produced trophic factors in the androgenic rescue of SNB motoneurons and further suggest that trophic factor interactions at the perineum play a crucial role in masculinization of this neural system.

Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the tis-11 primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of ob / ob mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in db / db mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR) and the signal-transducing beta-subunits gp130 and leukemia inhibitory factor receptor-beta (LIFR). CNTFR can function in either membrane-bound or soluble forms. The membrane-bound form mediates the neuronal actions of CNTF, whereas the soluble form serves to confer cytokine responsiveness to non-neuronal cells expressing gp130 and LIFR. The objective of this work was to analyze whether the two receptor isoforms differ in their ability to interact functionally with CNTF and related proteins. Two new types of CNTF variants, characterized by weakened interactions with either CNTFR or both LIFR and gp130, were developed, and the biological activities of these and other mutants were determined in non-neuronal versus neuronal cells, as well as in non-neuronal cells transfected with an expression vector for CNTFR. Membrane anchoring of CNTFR was found to render the CNTF receptor complex relatively insensitive to changes in agonist affinity for either alpha- or beta-receptor subunits and to promote a more efficient interaction with a gp130-depleting antagonistic variant of CNTF. As a result of this phenomenon, which can be rationalized in terms of the multivalent nature of CNTF receptor interaction, CNTF variants display striking changes in receptor selectivity.