Discovery and optimization of 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazines as novel,CNS penetrant pan-muscarinic antagonists

This letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M₄ antagonists, based on a 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazine core, identified from a high-throughput screening campaign. A multi-dimensional optimization effort enhanced potency at human M₄ (hM₄ IC₅₀s < 200 nM), with only moderate species differences noted, and with enantioselective inhibition. Moreover, CNS penetration proved attractive for this series (rat brain:plasma K_p = 2.1, K_p,uu = 1.1). Despite the absence of the prototypical mAChR antagonist basic or quaternary amine moiety, this series displayed pan-muscarinic antagonist activity across M_1-5 (with 9- to 16-fold functional selectivity at best). This series further expands the chemical diversity of mAChR antagonists.     

Multiplex gene removal by two-step polymerase chain reactions

Precise DNA manipulation is critical for molecular biotechnology. Restriction enzyme-based approaches are limited by their requirement of specific enzyme sites. Restriction-free cloning has greatly improved the flexibility and speed of precise DNA assembly. Most of these approaches focus on DNA assembly rather than gene removal. Here we present a polymerase chain reaction (PCR)-based cloning method that allows removal of multiple gene segments from plasmids without using restriction enzymes and thermostable ligase. We demonstrate simultaneous removal of three gene segments from a plasmid. This approach could be beneficial to DNA library construction, genetic and protein engineering, and synthetic biology.     

Bicyclic α,ω-dicarboxylic acid derivatives from a colonial tunicate of the family Polyclinidae

In the course of our search for bioactive metabolites from a colonial tunicate of the family Polyclinidae, six new (1–6) cyclic fatty acid derivatives were isolated. Their planar structures were established on the basis of NMR and MS spectroscopic analyses. The relative configuration was determined by NOESY experiment. Compounds 1–6 represent a fused bicyclic skeleton possibly derived from α,ω-dicarboxylic acids such as eicosanedioic acid or docosanedioic acid via a Diels–Alder type of cyclization. Compounds 1–4 and 6 showed mild cytotoxicity against a panel of five human solid tumor cell lines.     

The fermentation process for l-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli

Summary A process for l-phenylalanine production was studied using a tyrosine auxotrophic regulatory mutant of Escherichia coli, resistant to both -2-thienyl-dl-alanine and p-fluoro-dl-phenylalanine. Fermentations were carried out in a 30-1 fermentor with intermittent feeding of glucose plus phosphate. The mutant accumulated l-phenylalanine in the fermentation broth up to 15 g/l at pH 7.0 and 33°C. Column chromatography on a strong cation exchanger was employed as the most effective step in the purification of l-phenyl-alanine from the broth. This step brought about 4-fold concentration of the product with 96% recovery.     

Synthesis of oxidative metabolites of CRA13 and their analogs: Identification of CRA13 active metabolites and analogs thereof with selective CB2R affinity

CRA13; a peripheral dual CB₁R/CB₂R agonist with clinically proven analgesic properties, infiltrates into CNS producing adverse effects due to central CB₁R agonism. Such adverse effects might be circumvented by less lipophilic compounds with attenuated CB₁R affinity. Metabolism produces less lipophilic metabolites that might be active metabolites. Some CRA13 oxidative metabolites and their analogues were synthesized as less lipophilic CRA13 analogues. Probing their CB₁R and CB₂R activity revealed the alcohol metabolite 8c as a more potent and more effective CB₂R ligand with attenuated CB₁R affinity relative to CRA13. Also, the alcohol analogue 8b and methyl ester 12a possessed enhanced CB₂R affinity and reduced CB₁R affinity. The CB₂R binding affinity of alcohol analogue 8b was similar to CRA13 while that of methyl ester 12a was more potent. In silico study provided insights into the possible molecular interactions that might explain the difference in the elicited biological activity of these compounds.     

Relationship of the lung microbiome with PD-L1 expression and immunotherapy response in lung cancer

IL-35 subunit EBI3 is up-regulated in pulmonary fibrosis tissues. In this study, we investigated the pathological role of EBI3 in pulmonary fibrosis and dissected the underlying molecular mechanism. Bleomycin-induced pulmonary fibrosis mouse model was established, and samples were performed gene expression analyses through RNAseq, qRT-PCR and Western blot. Wild type and EBI3 knockout mice were exposed to bleomycin to investigate the pathological role of IL-35, via lung function and gene expression analyses. Primary lung epithelial cells were used to dissect the regulatory mechanism of EBI3 on STAT1/STAT4 and STAT3. IL-35 was elevated in both human and mouse with pulmonary fibrosis. EBI3 knockdown aggravated the symptoms of pulmonary fibrosis in mice. EBI3 deficiency enhanced the expressions of fibrotic and extracellular matrix-associated genes. Mechanistically, IL-35 activated STAT1 and STAT4, which in turn suppressed DNA enrichment of STAT3 and inhibited the fibrosis process. IL-35 might be one of the potential therapeutic targets for bleomycin-induced pulmonary fibrosis.