Design, Synthesis, and Development of Pyrazolo[1,5-a]pyrimidine Derivatives as a Novel Series of Selective PI3Kδ Inhibitors: Part II-Benzimidazole Derivatives.

Phosphoinositide 3-kinase (PI3K) is the family of lipid kinases participating in vital cellular processes such as cell proliferation, growth, migration, or cytokines production. Due to the high expression of these proteins in many human cells and their involvement in metabolism regulation, normal embryogenesis, or maintaining glucose homeostasis, the inhibition of PI3K (especially the first class which contains four subunits: α, ß, γ, δ) is considered to be a promising therapeutic strategy for the treatment of inflammatory and autoimmune diseases such as systemic lupus erythematosus (SLE) or multiple sclerosis. In this work, we synthesized a library of benzimidazole derivatives of pyrazolo[1,5-a]pyrimidine representing a collection of new, potent, active, and selective inhibitors of PI3Kδ, displaying IC50 values ranging from 1.892 to 0.018 µM. Among all compounds obtained, CPL302415 (6) showed the highest activity (IC50 value of 18 nM for PI3Kδ), good selectivity (for PI3Kδ relative to other PI3K isoforms: PI3Kα/δ = 79; PI3Kß/δ = 1415; PI3Kγ/δ = 939), and promising physicochemical properties. As a lead compound synthesized on a relatively large scale, this structure is considered a potential future candidate for clinical trials in SLE treatment.

A novel JAK/ROCK inhibitor, CPL409116, demonstrates potent efficacy in the mouse model of systemic lupus erythematosus.

Systemic lupus erythematosus is a chronic inflammatory disease, in which treatment is still limited due to suboptimal efficacy and toxicities associated with the available therapies. JAK kinases are well known to play an important role in systemic lupus erythematous. There is growing evidence that ROCK kinases are also important in disease development. In this paper, we present the results of the development of CPL409116, a dual JAK and ROCK inhibitor. The studies we performed demonstrate that this molecule is an effective JAK and ROCK inhibitor which efficiently blocks disease progression in NZBWF1/J mouse models of systemic lupus erythematous.

Significance of NF-kappaB signaling and PARP1 activity in the TNF-induced inhibition of PHEX gene expression in human osteoblasts.

Although loss of bone mineral density is a common symptom of chronic inflammatory diseases, its mechanisms are still poorly understood. The PHEX gene encodes a Zn-endopeptidase expressed in osteoblasts and contributes to bone mineralization. Data derived from rodents has indicated co-repression of the PHEX gene by the NF-κB pathway and poly(ADP-ribose) polymerase 1 (PARP1). The aim of this study was to determine the molecular mechanism involved in TNF-mediated downregulation of PHEX expression in human osteoblasts and human osteosarcoma cell line. We observed that activation of the NF-κB pathway by TNF was manifested as a nuclear increase in RELA and NFKB1 heterodimer. We found that TNF reduced PHEX expression and the proteasome inhibitor reversed this effect in osteosarcoma cell line. Contrary to the effects seen in rodents, inhibition of PARP1 enzymatic activity did not significantly reverse the effect of TNF on the human PHEX gene expression. EMSA studies showed that the number of adenines in the PHEX proximal promoter is crucial for the transcription factors' interactions within that region. The obtained results support the hypothesis indicating the existence of a molecular mechanism of gene repression that involves a poly adenine-rich region of the proximal gene promoters and PARP1 transcriptional activity.

Seasonal postembryonic maturation of the diurnal rhythm of serotonin in the chicken pineal gland.

Previously, we have demonstrated the postembryonic development of chicken (Gallus gallus domesticus L.) pineal gland functions expressed as changes in melatonin (MEL) biosynthesis. Pineal concentrations of MEL and its precursor serotonin (5-HT) were shown to increase between the 2nd and 16th day of life. We also found that levels of the mRNAs encoding the enzymes participating in the final two steps of MEL biosynthesis from 5-HT: arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), as well as their enzymatic activities, were raised during postembryonic development. Moreover, the manner of these changes was season-of-hatch dependent, even in animals kept under constant laboratory conditions (L:D 12:12). The most pronounced changes were seen in the concentrations of 5-HT and MEL, as well as in Aanat mRNA level and its enzymatic activity. The high daily variability in 5-HT content suggested that season- and age-dependent changes in the activity of the chicken pineal gland might rely on the availability of 5-HT, i.e. it may be limited by changes in pineal tryptophan (TRP) and/or 5-hydroxytryptophan (5-HTP) levels as well as by the activity of tryptophan hydroxylase (TPH) and aromatic l-amino acid decarboxylase (AADC): two enzymes participating in the conversion of TRP to 5-HT. The present study was undertaken with the following objectives: (1) to examine whether the pineal concentration of the 5-HT precursors TRP and 5-HTP exhibit age- and season-related changes; (2) to look for season-related differences in the transcription of the Tph1 and Ddc genes encoding enzymes TPH and AADC; (3) to identify the step(s) in postembryonic development in which these season-related variations in pineal gland function are most pronounced. Male Hy-line chickens hatched in the summer or winter, from eggs laid by hens held in L:D 16:8 conditions were kept from the day of hatch in L:D 12:12 conditions. At the age of 2 or 9 days, animals were sacrificed every 2 or 4 h over a 24-h period and their pineal glands were isolated under dim red light and processed for the measurement of (i) the pineal content of TRP, 5-HTP and 5-HT, and (ii) the level of Tph1 and Ddc mRNAs. Circadian rhythmicity of all the measured parameters was evaluated by the cosinor method. The pineal levels of TRP and 5-HT as well as the Tph1 and Ddc transcripts changed during postembryonic development in a season-related way. Whereas, the 5-HTP concentration did not vary between animals from both age groups, regardless of the season. Circadian rhythmicity of all the measured parameters was dependent on both the age and the season of hatch, and was greatest in older animals in the summer. These findings indicated that the efficiency of season-related MEL biosynthesis, reported previously, is limited by 5-HT availability and this limitation depends on the transcription of both the Tph1 and Ddc genes. Moreover, Ddc mRNA level in 9-d-old birds changed rhythmically, even though this gene is generally considered to be arrhythmic.

Effects of period RNAi on V-ATPase expression and rhythmic pH changes in the vas deferens of Spodoptera littoralis (Lepidoptera: Noctuidae).

Circadian clocks (oscillators) regulate multiple aspects of insect behaviour and physiology. The circadian system located in the male reproductive tract of Lepidoptera orchestrates rhythmic sperm release from testis and sperm maturation in the upper vas deferens (UVD). Our previous research on the cotton leafworm, Spodoptera littoralis, suggested rhythmic changes in the V-ATPase levels in the UVD epithelium, which correlated with rhythmic pH fluctuations in the UVD lumen. However, it was not known whether UVD cells contain clock mechanism that generates these daily fluctuations. In the current paper, we show circadian rhythm in the expression of clock gene period at the mRNA and protein level in the UVD epithelium. To determine the role of PER in V-ATPase and pH regulation, testes-UVD complexes were treated in vitro with double-stranded fragments of per mRNA (dsRNA). This treatment, which transiently lowered per mRNA and protein in the UVD, altered expression of V-ATPase c subunit. In addition, per RNAi caused a significant delay in the UVD lumen acidification. These data demonstrate that the UVD molecular oscillator involving the period gene plays an essential role in the regulation of rhythmic V-ATPase activity and periodic acidification of the UVD lumen.

Pineal arylalkylamine N-acetyltransferase (Aanat) gene expression as a target of inflammatory mediators in the chicken.

Previously, we demonstrated that experimental peritonitis in chickens was attenuated by treatment with exogenous melatonin, while the developing inflammation decreased pineal AANAT activity. This suggested the existence of a bidirectional relationship between the activated immune system and pineal gland function. The aim of the present study was to identify the step(s) in the chicken pineal melatonin biosynthetic pathway that are affected by inflammation. Peritonitis was evoked by i.p. injection of thioglycollate solution, either 2h after the start, or 2h before the end of the light period, and the animals were sacrificed 4h later. The effect of inflammation on the expression of genes encoding enzymes participating in melatonin biosynthesis in the pineal gland, i.e. tryptophan hydroxylase 1 (Tph1), dopa decarboxylase (Ddc), arylalkylamine N-acetyltransferase (Aanat) and acetylserotonin O-methyltransferase (Asmt), was evaluated by qPCR. The pineal and serum melatonin concentration as well as the content of its precursors in the pineal gland were measured, along with the activity of the relevant biosynthetic enzymes. Developing peritonitis caused an increase in the pineal levels of the Tph1 mRNA during the night and the Asmt mRNA during the day, while nocturnal Aanat transcription was reduced. Both the pineal and serum melatonin level and the pineal content of N-acetylserotonin (NAS) were decreased during the night in birds with peritonitis. The amount and activity of pineal AANAT were significantly reduced, while the activity of HIOMT was increased under these experimental conditions. These results indicate that the observed decrease in MEL biosynthesis in chickens with developing inflammation is a result of transcriptional downregulation of the Aanat gene, followed by reduced synthesis and activity of the encoded enzyme.