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Investigation of cationicity and structure of pseudin-2 analogues for enhanced bacterial selectivity and anti-inflammatory activity.

ABSTRACT: Pseudin-2 (Ps), isolated from the frog Pseudis paradoxa, exhibits potent antibacterial activity and cytotoxicity. To develop antimicrobial peptides with anti-inflammatory activity and low cytotoxicity, we designed Ps analogues with Lys substitutions, resulting in elevated amphipathic ?-helical structure and cationicity. We further substituted Gly11 with Pro (Ps-P analogues) to increase bacterial cell selectivity. Ps analogues retained antimicrobial activity and exhibited reduced cytotoxicity, whereas Ps-P analogues exhibited lower cytotoxicity and antimicrobial activity. Tertiary structures revealed that Ps has a linear ?-helix from Leu2 to Glu24, whereas Ps-P has a bend at Pro11 between two short ?-helixes. Using various biophysical experiments, we found that Ps analogues produced much higher membrane depolarization than Ps-P analogues, whereas Ps-P analogues may penetrate bacterial cell membranes. Ps and its analogue Ps-K18 exhibited potent anti-inflammatory activity in LPS-stimulated RAW264.7 and mouse dendritic cells via a mechanism involving the Toll-like receptor 4 (TLR4) pathway. These activities may arise from their direct inhibition of the formation of TLR4-MD-2_LPS complex, implying that amphipathic ?-helical structure with an optimum balance between enhanced cationicity and hydrophobicity may be essential for their anti-inflammatory activity. The bent structure provided by Pro substitution plays an important role in enhancing bacterial cell selectivity and cell penetration.

SUBMITTER: Jeon D

PROVIDER: S-EPMC5431190 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Investigation of cationicity and structure of pseudin-2 analogues for enhanced bacterial selectivity and anti-inflammatory activity.

Jeon Dasom D Jeong Min-Cheol MC Jacob Binu B Bang Jeong Kyu JK Kim Eun-Hee EH Cheong Chaejoon C Jung In Duk ID Park Yoonkyung Y Kim Yangmee Y

Scientific reports 20170503 1

Pseudin-2 (Ps), isolated from the frog Pseudis paradoxa, exhibits potent antibacterial activity and cytotoxicity. To develop antimicrobial peptides with anti-inflammatory activity and low cytotoxicity, we designed Ps analogues with Lys substitutions, resulting in elevated amphipathic α-helical structure and cationicity. We further substituted Gly<sup>11</sup> with Pro (Ps-P analogues) to increase bacterial cell selectivity. Ps analogues retained antimicrobial activity and exhibited reduced cytot ...[more]

PMID: 28469145

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Project description:Background: Excessive inflammation results in severe tissue damage as well as serious acute or chronic disorders, and extensive research has focused on finding new anti-inflammatory hit compounds with safety and efficacy profiles from natural products. As promising therapeutic entities for the treatment of inflammation-related diseases, fusaproliferin and its analogs have attracted great interest. However, the underlying anti-inflammatory mechanism is still poorly understood and deserves to be further investigated. Methods: For the estimation of the anti-inflammatory activity of fusaproliferin (1) and its analogs (2-4) in vitro and in vivo, lipopolysaccharide (LPS)-induced RAW264.7 macrophages and zebrafish embryos were employed. Then, transcriptome analysis was applied to guide subsequent western blot analysis of critical proteins in related signaling pathways. Surface plasmon resonance assays (SPR) combined with molecular docking analyses were finally applied to evaluate the affinity interactions between 1-4 and TLR4 and provide a possible interpretation of the downregulation of related signaling pathways. Results: 1-4 significantly attenuated the production of inflammatory messengers, including nitric oxide (NO), reactive oxygen species (ROS), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), as well as nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in LPS-induced RAW264.7 macrophages. Transcriptome analyses based on RNA-seq indicated the ability of compound 1 to reverse LPS stimulation and the nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPKs) signaling pathways contribute to the anti-inflammatory process. Experimental verification at the protein level revealed that 1 can inhibit the activation of inhibitor of NF-κB kinase (IKK), degradation of inhibitor of NF-κB (IκB), and phosphorylation of NF-κB and reduce nuclear translocation of NF-κB. 1 also decreased the phosphorylation of MAPKs, including p38, extracellular regulated protein kinases (ERK), and c-Jun N-terminal kinase (JNK). SPR assays and molecular docking results indicated that 1-4 exhibited affinity for the TLR4 protein with KD values of 23.5-29.3 μM. Conclusion: Fusaproliferin and its analogs can be hit compounds for the treatment of inflammation-associated diseases.

Project description:The 5-HT2A receptor is thought to be the primary target for psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) and other serotonergic hallucinogens (psychedelic drugs). Although a large amount of experimental work has been conducted to characterize the pharmacology of psilocybin and its dephosphorylated metabolite psilocin (4-hydroxy-N,N-dimethyltryptamine), there has been little systematic investigation of the structure-activity relationships (SAR) of 4-substituted tryptamine derivatives. In addition, structural analogs of psilocybin containing a 4-acetoxy group, such as 4-acetoxy-N,N-dimethyltryptamine (4-AcO-DMT), have appeared as new designer drugs, but almost nothing is known about their pharmacological effects. To address the gap of information, studies were conducted with 17 tryptamines containing a variety of symmetrical and asymmetrical N,N-dialkyl substituents and either a 4-hydroxy or 4-acetoxy group. Calcium mobilization assays were conducted to assess functional activity at human and mouse 5-HT2 subtypes. Head-twitch response (HTR) studies were conducted in C57BL/6J mice to assess 5-HT2A activation in vivo. All of the compounds acted as full or partial agonists at 5-HT2 subtypes, displaying similar potencies at 5-HT2A and 5-HT2B receptors, but some tryptamines with bulkier N-alkyl groups had lower potency at 5-HT2C receptors and higher 5-HT2B receptor efficacy. In addition, O-acetylation reduced the in vitro 5-HT2A potency of 4-hydroxy-N,N-dialkyltryptamines by about 10- to 20-fold but did not alter agonist efficacy. All of the compounds induce head twitches in mice, consistent with an LSD-like behavioral profile. In contrast to the functional data, acetylation of the 4-hydroxy group had little effect on HTR potency, suggesting that O-acetylated tryptamines may be deacetylated in vivo, acting as prodrugs. In summary, the tryptamine derivatives have psilocybin-like pharmacological properties, supporting their classification as psychedelic drugs.

Dataset Information

Investigation of cationicity and structure of pseudin-2 analogues for enhanced bacterial selectivity and anti-inflammatory activity.

Publications

Investigation of cationicity and structure of pseudin-2 analogues for enhanced bacterial selectivity and anti-inflammatory activity.

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