Discovery and Binding Mechanism of Pyrazoloisoquinoline-Based Novel ß-Arrestin Inverse Agonists of the Kappa-Opioid Receptor.

Chronic exposure to stress or unwanted stimuli has been known to activate kappa opioid receptor/dynorphin (KOR/DYN) systems, which could induce depressive states and develop into some psychiatric disorders. Here, we report the first discovery of pyrazoloisoquinoline-based novel KOR ß-arrestin inverse agonists through synthesis, structure-activity relationships, optimization, and the biological evaluations of µ/κ/δ opioid receptor activities with cAMP and ß-arrestin recruitment assays. The optimized compound 7q shows potent and selective ß-arrestin inverse agonism at KOR with an EC50 value of 9.33 nM in contrast to lower activities at DOR and no activity at MOR. Moreover, we use molecular dynamics simulations to predict the binding mode of the inverse agonist and propose a mechanism for the inverse agonism. We find that the transmembrane helix 6 position of the activated state is different for the OR subtypes, leading to significantly different interactions between the receptor and ß-arrestin.

Discovery of pyrazole-1-carboxamide derivatives as novel Gi-biased µ-opioid receptor agonists.

µ-Opioid receptor (MOR) Gi-biased agonists with no recruitment of ß-arrestin were introduced as a new analgesic strategy to overcome the conventional undesirable side effects of opioid receptor-targeted drugs, such as tolerance, addiction, respiratory depression, and constipation. For the development of novel Gi-biased MOR agonists, the design, synthesis, and structure-activity relationship (SAR) analysis of the aminopyrazole core skeleton were conducted according to the current SAR data of PZM21 (2a) and its derivatives. New derivatives were biologically evaluated for their agonistic effects on cyclic adenosine monophosphate (cAMP) levels for the Gi pathway and ß-arrestin recruitment in MOR/κ-opioid receptor/δ opioid receptor. An optimized selective Gi-biased agonist, Compound 17a, was discovered with potent cAMP inhibitory activities, with a 50% efficacy concentration value of 87.1 nM and no activity in the MOR ß-arrestin pathway and other subtypes. The in vivo pain relief efficacy of Compound 17a was confirmed in a dose-dependent manner with spinal nerve ligation and cisplatin-induced peripheral neuropathy rodent neuropathic pain models.

Genome-based analysis for the bioactive potential of Streptomyces yeochonensis CN732, an acidophilic filamentous soil actinobacterium.

BACKGROUND: Acidophilic members of the genus Streptomyces can be a good source for novel secondary metabolites and degradative enzymes of biopolymers. In this study, a genome-based approach on Streptomyces yeochonensis CN732, a representative neutrotolerant acidophilic streptomycete, was employed to examine the biosynthetic as well as enzymatic potential, and also presence of any genetic tools for adaptation in acidic environment. RESULTS: A high quality draft genome (7.8 Mb) of S. yeochonensis CN732 was obtained with a G + C content of 73.53% and 6549 protein coding genes. The in silico analysis predicted presence of multiple biosynthetic gene clusters (BGCs), which showed similarity with those for antimicrobial, anticancer or antiparasitic compounds. However, the low levels of similarity with known BGCs for most cases suggested novelty of the metabolites from those predicted gene clusters. The production of various novel metabolites was also confirmed from the combined high performance liquid chromatography-mass spectrometry analysis. Through comparative genome analysis with related Streptomyces species, genes specific to strain CN732 and also those specific to neutrotolerant acidophilic species could be identified, which showed that genes for metabolism in diverse environment were enriched among acidophilic species. In addition, the presence of strain specific genes for carbohydrate active enzymes (CAZyme) along with many other singletons indicated uniqueness of the genetic makeup of strain CN732. The presence of cysteine transpeptidases (sortases) among the BGCs was also observed from this study, which implies their putative roles in the biosynthesis of secondary metabolites. CONCLUSIONS: This study highlights the bioactive potential of strain CN732, an acidophilic streptomycete with regard to secondary metabolite production and biodegradation potential using genomics based approach. The comparative genome analysis revealed genes specific to CN732 and also those among acidophilic species, which could give some insights into the adaptation of microbial life in acidic environment.