Spectroscopic view on the interaction between the psoralen derivative amotosalen and DNA.

Psoralens are eponymous for PUVA (psoralen plus UV-A radiation) therapy, which inter alia can be used to treat various skin diseases. Based on the same underlying mechanism of action, the synthetic psoralen amotosalen (AMO) is utilized in the pathogen reduction technology of the INTERCEPT® Blood System to inactivate pathogens in plasma and platelet components. The photophysical behavior of AMO in the absence of DNA is remarkably similar to that of the recently studied psoralen 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT). By means of steady-state and time-resolved spectroscopy, intercalation and photochemistry of AMO and synthetic DNA were studied. AMO intercalates with a higher affinity into A,T-only DNA (KD = 8.9 × 10-5 M) than into G,C-only DNA (KD = 6.9 × 10-4 M). AMO covalently photobinds to A,T-only DNA with a reaction quantum yield of ΦR = 0.11. Like AMT, it does not photoreact following intercalation into G,C-only DNA. Femto- and nanosecond transient absorption spectroscopy reveals the characteristic pattern of photobinding to A,T-only DNA. For AMO and G,C-only DNA, signatures of a photoinduced electron transfer are recorded.

Development of an RNA-protein crosslinker to capture protein interactions with diverse RNA structures in cells.

Characterization of RNA-protein interaction is fundamental for understanding the metabolism and function of RNA. UV crosslinking has been widely used to map the targets of RNA-binding proteins, but is limited by low efficiency, requirement for zero-distance contact, and biases for single-stranded RNA structure and certain residues of RNA and protein. Here, we report the development of an RNA-protein crosslinker (AMT-NHS) composed of a psoralen derivative and an N-hydroxysuccinimide ester group, which react with RNA bases and primary amines of protein, respectively. We show that AMT-NHS can penetrate into living yeast cells and crosslink Cbf5 to H/ACA snoRNAs with high specificity. The crosslinker induced different crosslinking patterns than UV and targeted both single- and double-stranded regions of RNA. The crosslinker provides a new tool to capture diverse RNA-protein interactions in cells.

pH-Dependent Photoinduced Interconversion of Furocoumaric and Furocoumarinic Acids.

Photo-controlled or photo-regulated molecules, especially biologically active and operating in physiological conditions, are in steady demand. Herein, furocoumaric and furocoumarinic acids being (Z/E)-isomers relative to each other were obtained in two stages starting from psoralen: the alkaline solvolysis of psoralen led to furocoumaric acid, which was further Z → E photoisomerized (365 nm) to furocoumarinic acid. The kinetics of Z → E photoisomerization was monitored by HPLC and UV-vis spectrophotometry. Photophysical characteristics in the aqueous phase for both acids, as well as the reversibility of (Z/E) photoisomerization process, were also assessed. Furocoumarinic acid was found to be visibly fluorescent at pH 2.0-12.0, with the maxima of fluorescence emission spectra being pH-dependent. The reverse E → Z photoisomerization predicted by quantum chemistry calculations as energetically favorable for the monoanionic form of furocoumarinic acid was proved in the experiment while being complicated by pyrone ring closure back to psoralen in acidic and neutral conditions. The preparative synthesis of furocoumarinic acid outlined in this work is particularly valuable in view of a wide range of pharmacological effects previously predicted for this compound.

Botulinum neurotoxin inhibitor binding dynamics and kinetics relevant for drug design.

BACKGROUND: A natural product analog, 3-(4-nitrophenyl)-7H-furo[3,2-g]chromen-7-one, which is a nitrophenyl psoralen (NPP) was found to be an effective inhibitor of botulinum neurotoxin type A (BoNT/A). METHODS: In this work, we performed enzyme inhibition kinetics and employed biochemical techniques such as isothermal calorimetry (ITC) and fluorescence spectroscopy as well as molecular modeling to examine the kinetics and binding mechanism of NPP inhibitor with BoNT/A LC. RESULTS: Studies of inhibition mechanism and binding dynamics of NPP to BoNT/A light chain (BoNT/A LC) showed that NPP is a mixed type inhibitor for the zinc endopeptidase activity, implying that at least part of the inhibitor-enzyme binding site may be different from the substrate-enzyme binding site. By using biochemical techniques, we demonstrated NPP forms a stable complex with BoNT/A LC. These observations were confirmed by Molecular Dynamics (MD) simulation, which demonstrates that NPP binds to the site near the active site. CONCLUSION: The NPP binding interferes with BoNT/A LC binding to the SNAP-25, hence, inhibits its cleavage. Based on these results, we propose a modified strategy for designing a molecule to enhance the efficiency of the inhibition against the neurotoxic effect of BoNT. GENERAL SIGNIFICANCE: Insights into the interactions of NPP with BoNT/A LC using biochemical and computational approaches will aid in the future development of effective countermeasures and better pharmacological strategies against botulism.

Selective inhibition of carbonic anhydrase IX and XII by coumarin and psoralen derivatives.

A small library of coumarin and their psoralen analogues EMAC10157a-b-d-g and EMAC10160a-b-d-g has been designed and synthesised to investigate the effect of structural modifications on their inhibition ability and selectivity profile towards carbonic anhydrase isoforms I, II, IX, and XII. None of the new compounds exhibited activity towards hCA I and II isozymes. Conversely, both coumarin and psoralen derivatives were active against tumour associated isoforms IX and XII in the low micromolar or nanomolar range of concentration. These data further corroborate our previous findings on analogous derivatives, confirming that both coumarins and psoralens are interesting scaffolds for the design of isozyme selective hCA inhibitors.

Structural modularity of the XIST ribonucleoprotein complex.

Long noncoding RNAs are thought to regulate gene expression by organizing protein complexes through unclear mechanisms. XIST controls the inactivation of an entire X chromosome in female placental mammals. Here we develop and integrate several orthogonal structure-interaction methods to demonstrate that XIST RNA-protein complex folds into an evolutionarily conserved modular architecture. Chimeric RNAs and clustered protein binding in fRIP and eCLIP experiments align with long-range RNA secondary structure, revealing discrete XIST domains that interact with distinct sets of effector proteins. CRISPR-Cas9-mediated permutation of the Xist A-repeat location shows that A-repeat serves as a nucleation center for multiple Xist-associated proteins and m6A modification. Thus modular architecture plays an essential role, in addition to sequence motifs, in determining the specificity of RBP binding and m6A modification. Together, this work builds a comprehensive structure-function model for the XIST RNA-protein complex, and suggests a general strategy for mechanistic studies of large ribonucleoprotein assemblies.

Ultrasonic-enhanced surface-active ionic liquid-based extraction and defoaming for the extraction of psoralen and isopsoralen from Psoralea corylifolia seeds.

Recently, integrated and sustainable methods for extracting active substances from plant materials using green solvents, i.e., ionic liquids, have gained increasing attention. Ionic liquids showsuperiority over conventional organic solvents; however, they also exhibit negative factors and problems, such as high viscosity, poor water intermiscibility, intensive foaming and poor affinity for fat-soluble substances. The proposed method utilizes ultrasonic-enhanced surface-active ionic liquid-based extraction and defoaming (UESILED) to improve the extraction efficiency of ionic liquids. Single-factor experiments and a Box-Behnken design (BBD) were utilized to optimize the extraction procedure. The optimal conditions were as follows: extraction solvent, [C10MIM]Br; ultrasonic treatment time, 28 min; ultrasonic irradiation power, 437 W; liquid-solid ratio, 10 mL/g; particle size, 60 ~ 80 mesh; ultrasonication temperature, 313 K; and [C10MIM]Br solution concentration, 0.5 mol/L. In comparison with those of other reference extraction methods, the proposed method exhibited higher yields of two furocoumarins and operational feasibility. Moreover, the mechanism of UESILED was elaborated in terms of accelerating infiltration, dissolution and defoaming. The feasible and efficient ultrasonic-enhanced ionic liquid-based extraction established in this study strongly contributes to overcoming the limitations of ionic liquid solvents. The present research indicates that this improved process will be beneficial for the extraction of other fat-soluble substances and provides promising concepts and experimental data.

Psoralen as an interstrand DNA crosslinker in the selection of DNA-Encoded dynamic chemical library.

DNA-encoded chemical library (DEL) has emerged as a powerful technology for ligand discovery in biomedical research. Recently, we have developed a DNA-encoded dynamic library (DEDL) approach by incorporating the concept of dynamic combinatorial library (DCL) with DELs. DEDL has shown excellent potential in ligand discovery towards a variety of protein targets. However, the requirement of having a pair of unnatural p-stilbazoles as the interstrand DNA crosslinker has limited the chemical diversity of DEDLs. Here, we replaced p-stilbazole with psoralen (PS) and tested the feasibility of psoralen as the crosslinker in DEDL selection. Since psoralen is commercially available and does not require any special crosslinking partner, existing DELs may be directly used to create high-diversity DEDLs. This study is expected to greatly facilitate the development of DEDLs as a versatile tool in drug discovery.

Psoralen: A Biologically Important Coumarin with Emerging Applications.

Coumarin belongs to a class of lactones that are fundamentally comprised of a benzene ring fused to an α-pyrone ring; these lactones are known as benzopyrones. Similarly, coumarin has a conjugated electron-rich framework and good charge-transport properties. Plants produce coumarin as a chemical response to protect themselves from predation. Coumarins are used in different products, such as cosmetics, additives, perfumes, aroma enhancers in various tobaccos and some alcoholic drinks, and they play a relevant role in natural products and in organic and medicinal chemistry. In addition, as candidate drugs, many coumarin compounds have strong pharmacological activity, low toxicity, high bioavailability and better curative effects and have been used to treat various types of diseases. Various endeavors were made to create coumarin-based anticoagulant, antimicrobial, antioxidant, anticancer, antidiabetic, antineurodegenerative, analgesic and anti-inflammatory agents. A class of chemical compounds called furocoumarins has phototoxic properties and is naturally synthesized via the fusion of coumarin to a furan ring in different plant species. Psoralens belong to the furocoumarin class and occur naturally in various plants, e.g., lemons, limes, and parsnips. Angelicin is an isomer of psoralens, and most furocoumarins, e.g., xanthotoxin, bergapten, and nodekenetin, are derivatives of psoralens or angelicin. The present work demonstrated that psoralen molecules exhibit anti-tumoral activity against breast cancer and influence different intracellular signals to maintain the high survival of breast cancer cells. Psoralens perform different functions, e.g., antagonize metabolic pathways, protease enzymes, and cell cycle progression and even interfere in the crosslinking between receptors and growth factor mitogenic signaling.

Psoralen Derivatives as Inhibitors of Mycobacterium tuberculosis Proteasome.

Protein degradation is a fundamental process in all living organisms. An important part of this system is a multisubunit, barrel-shaped protease complex called the proteasome. This enzyme is directly responsible for the proteolysis of ubiquitin- or pup-tagged proteins to smaller peptides. In this study, we present a series of 92 psoralen derivatives, of which 15 displayed inhibitory potency against the Mycobacterium tuberculosis proteasome in low micromolar concentrations. The best inhibitors, i.e., 8, 11, 13 and 15, exhibited a mixed type of inhibition and overall good inhibitory potency in biochemical assays. N-(cyanomethyl)acetamide 8 (Ki = 5.6 µM) and carboxaldehyde-based derivative 15 (Ki = 14.9 µM) were shown to be reversible inhibitors of the enzyme. On the other hand, pyrrolidine-2,5-dione esters 11 and 13 irreversibly inhibited the enzyme with Ki values of 4.2 µM and 1.1 µM, respectively. In addition, we showed that an established immunoproteasome inhibitor, PR-957, is a noncompetitive irreversible inhibitor of the mycobacterial proteasome (Ki = 5.2 ± 1.9 µM, kinact/Ki = 96 ± 41 M-1·s-1). These compounds represent interesting hit compounds for further optimization in the development of new drugs for the treatment of tuberculosis.

Binding characteristics of aflatoxin B1 with free DNA in vitro.

In this paper, the binding characteristics of aflatoxin B1 (AFB1) with the herring sperm deoxyribonucleic acid (DNA) in vitro were investigated through different analytical methods. The ultraviolet-visible spectroscopy (UV-vis), fluorescence, and circular dichroism (CD) spectra results showed that a new AFB1-DNA complex was formed. All the results suggested that AFB1 interacted with free DNA in vitro in an intercalating binding mode. The results of the DNA melting experiments also showed that the melting temperature of DNA increased by about 12.1 °C due to the addition of AFB1, which was supposed to be closely related to the intercalation of AFB1 into DNA. The agar gel electrophoresis experiments further confirmed that the binding mode of AFB1 and free DNA in vitro was indeed intercalation. In addition, the fluorescence quenching induced by adding AFB1 to the ethidium bromide-DNA (EB-DNA) mixture indicated the presence of competitive non-covalent intercalating binding interaction with a competitive binding constant of 5.58 L/mol between AFB1, EB, and DNA. The thermodynamic data demonstrated that the main driving forces of the binding reaction were van der Waals forces and hydrogen bond. The resonance light scattering (RLS) assay results showed that the DNA binding saturation values of AFB1, EB, psoralen (PSO), and angelicin (ANG) were 2.14, 15.59, 0.74, and 0.74, respectively. These results indicated that the DNA binding capacity of AFB1 was weaker than that of EB, but stronger than those of PSO and ANG.

Psoralen Derivatives: Recent Advances of Synthetic Strategy and Pharmacological Properties.

Psoralen or furocoumarin is a linear three ring heterocyclic compound. Psoralens are planar, tricyclic compounds, consisting of a furan ring fused to a coumarin moiety. Psoralen has been known for a wide spectrum of biological activities, spanning from cytotoxic, photosensitizing, insecticidal, antibacterial to antifungal effect. Thus, several structural changes were introduced to explore the role of specific positions with respect to the biological activity. Convenient approaches utilized for the synthesis of psoralen skeleton can be categorized into two parts: (i) the preparation of the tricyclic ring system from resorcinol, (ii) the exocyclic modification of the intact ring system. Furthermore, although psoralens have been used in diverse ways, we mainly focus in this work on their clinical utility for the treatment of psioraisis, vitiligo and skin-related disorder.

Transformation of Psoralen and Isopsoralen by Human Intestinal Microbial In Vitro, and the Biological Activities of Its Metabolites.

Psoralen (P) and isopsoralen (IP) are the main active ingredients in the dried fruit of Psoralen corylifolia L. (PC), with a wide range of pharmacology activities. The intestinal bacteria biotransformation plays a central role in the metabolism of the complex ingredients in traditional Chinese medicine (TCM). Our study aimed to investigated the metabolic profile of P and IP in the intestinal condition, co-cultured with human fecal bacteria anaerobically. Four bio-transforming products were obtained, including 6,7-furano-hydrocoumaric acid (P-1) and 6,7-furano-hydro- coumaric acid methyl ester (P-2), which transformed from P, and 5,6-furano-hydrocoumaric acid (IP-1) and 5,6-furano-hydrocoumaric acid methyl ester (IP-2), which were transformed from IP. It is worth mentioning that IP-2 is a new compound that has not been published. Their structures were analyzed based on their spectroscopic data. Moreover, a highly sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was used to characterize the metabolic pathways of P, IP, and their bio-transforming products in the reaction samples. In addition, the dampening effects against the oxidative stress of P, IP, and their bio-transforming products by human intestinal flora were estimated in vitro via the human colorectal cells (HCT116) and heterogeneous human epithelial colorectal adenocarcinoma cells (Caco-2) cell lines. The results showed that the metabolites have stronger activity than P and IP, which possibly provides a basis for elucidating the treating mechanisms of PC extract against inflammatory bowel disease.

miR­488 negatively regulates osteogenic differentiation of bone marrow mesenchymal stem cells induced by psoralen by targeting Runx2.

It has been previously reported that psoralen, one of the active ingredients in Psoralea corylifolia, could induce osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), suggesting its potential to treat osteoporosis. Additionally, runt­related transcription factor 2 (Runx2) is a transcription factor that plays vital roles in BMSC osteogenic differentiation. However, whether and how microRNAs (miRNAs/miRs) modulate osteogenic differentiation induced by psoralen have not yet been examined, to the best of the authors' knowledge. The present study aimed to identify the miRNA target genes that regulate osteogenic differentiation of BMSCs induced by psoralen. A Cell Counting Kit­8 assay and alizarin red staining were used to detect the viability and osteogenic differentiation of BMSCs, respectively, under treatment with psoralen. miRNA microarray analysis was performed to identify the differentially expressed miRNAs under treatment with psoralen. A bioinformatics analysis and a luciferase reporter assay were conducted to identify the targets of miR­488. Finally, the mechanisms of miR­488 in psoralen­induced BMSC osteogenic differentiation were investigated using overexpression or inhibition methods in vitro. Cell viability was elevated and osteogenic differentiation of BMSCs was improved under treatment with psoralen. miRNA microarray analysis and further validation by reverse transcription­quantitative PCR revealed that miR­488 was downregulated during psoralen­induced BMSC osteogenic differentiation. Bioinformatics analysis and experimental validation by a luciferase reporter assay identified Runx2 as a potential target of miR­488. Overexpression of miR­488 by transfection with miR­488 mimics markedly inhibited the expression of Runx2, Osterix and alkaline phosphatase, whereas, the inhibition of miR­488 expression by the miR­488 inhibitor promoted their expression compared with the control. Rescue assays demonstrated that Runx2 overexpression partially rescued the inhibitory effect of miR­488 on BMSC osteogenic differentiation. The present results suggested that miR­488 is a negative regulator of psoralen­induced BMSC osteogenic differentiation by targeting Runx2, providing a possible therapeutic target for osteoporosis.

Furocoumarin Content of Fennel-Below the Safety Threshold.

Furocoumarins are known for their phototoxic and potential carcinogenic effects. These types of compounds have previously been reported from fennel (Foeniculum vulgare Mill.), a widely used medicinal plant and spice; however, no reliable quantitative data are available on the occurrence of these compounds in fennel fruits. For the first time, we report a comprehensive analysis of fennel fruit samples of different origins, representing a wide range of accessions for their furocoumarin content. Psoralene, 5-methoxypsoralene (bergapten), and imperatorin contents of 33 fennel samples were analyzed using a sensitive liquid chromatography-mass spectrometry (LC-MS) method. When applied at the highest therapeutic dose described in the monograph issued by the European Medicines Agency, the furocoumarin content of the fruits ranged up to 1.22 µg/d, which is below the most restrictive recommendations. Based on our findings, fennel consumption can be considered as safe, at least based on its low furocoumarin content.

Psoralen inhibits malignant proliferation and induces apoptosis through triggering endoplasmic reticulum stress in human SMMC7721 hepatoma cells.

BACKGROUND: Psoralen is a coumarin-like and coumarin-related benzofuran glycoside, which is a commonly used traditional Chinese medicine to treat patients with kidney and spleen-yang deficiency symptom. Psoralen has been reported to show estrogen-like activity, antioxidant activity, osteoblastic proliferation accelerating activity, antitumor effects and antibacterial activity. However, the antitumor mechanism of psoralen is not fully understood. This study aimed to investigate the therapeutic efficacy of psoralen in human hepatoma cell line SMMC7721 and the mechanism of antitumor effects. RESULTS: Psoralen inhibited proliferation of SMMC7721 in a dose- and time-dependent manner, and promoted apoptosis. Further, psoralen activated the ER stress signal pathway, including the expansion of endoplasmic reticulum, increasing the mRNA levels of GRP78, DDIT3, ATF4, XBP1, GADD34 and the protein levels of GDF15, GRP78, IRE1α, XBP-1s in a time-dependent manner. Psoralen induces cell cycle arrest at G1 phase by enhancing CyclinD1 and reducing CyclinE1 expression. Moreover, TUDC couldn't inhibit the psoralen-induced ER stress in SMMC7721 cells. CONCLUSIONS: Psoralen can inhibit the proliferation of SMMC7721 cells and induce ER stress response to induce cell apoptosis, suggesting that psoralen may represent a novel therapeutic option for the prevention and treatment hepatocellular carcinoma.

Polymer-lipid hybrid nanoparticles: A novel drug delivery system for enhancing the activity of Psoralen against breast cancer.

A polymer-lipid hybrid nanocarrier was developed to encapsulate psoralen (PSO) to improve its water solubility and bioavailability. The effects of PSO-loaded polymer-lipid hybrid nanoparticles (PSO-PLNs) on breast cancer MCF-7 cells were investigated. PSO-PLNs were prepared through a nanoprecipitation method and were optimized by a central composite design-response surface methodology using particle size and entrapment efficiency as indices. Dynamic light scattering and transmission electron microscopy analysis confirmed the physicochemical characterizations of PSO-PLNs, which had an average size of 93.44 ±â€¯2.39 nm and a zeta potential of -27.63 ±â€¯0.31 mV. In vitro drug release of PSO-PLNs was evaluated using dialysis and showed a delayed release compared with free PSO. The in vivo anticancer efficiency of PSO-PLNs was appreciated using a MCF-7 breast tumor model. Administration of PSO-PLNs showed similar antitumor efficacy but lower toxicity compared with doxorubicin. Our designed nanocarriers successfully optimized the pharmacokinetics of PSO via improved systemic delivery.

Photo-induced DNA interstrand cross-links formed by a coumarin-modified nucleoside.

Coumarins are a class of naturally occurring compounds that have been shown to form photochemical DNA interstrand cross-links (ICLs). However, study of a coumarin base has not been explored. Using nucleophilic substitution and phosphoramidite chemistry, we synthesized a coumarin base-containing oligonucleotide. Upon exposure to long-wave ultraviolet light, the coumarin-modified oligonucleotide formed ICLs with complementary oligonucleotide containing dT and dC opposite the coumarin base, presumably through a [2 + 2] cycloaddition mechanism. Moderate yields with both bases were observed; though, dT has a higher reactivity than dC. Overall, this work provides new means for biochemical characterization of ICLs formed by coumarins.

Strong Inhibitory Effects of Antisense Probes on Gene Expression through Ultrafast RNA Photocrosslinking.

We have reported the photochemical regulation of the intracellular antisense effect of antisense probes containing a photo-responsive artificial nucleic acid, 3-cyanovinylcarbazole nucleoside (CNV K). Here we focus on the importance of the photocrosslinking rate on the inhibitory effect on gene expression using photocrosslinkable antisense probes (pcASOs). The inhibitory effect of pcASOs on GFP gene expression was dependent on the photocrosslinking rate of 3-cyanovinylcarbazole with d-threoninol (CNV D), CNV K, or psoralen. The ultrafast RNA photocrosslinking induced the formation of a thermally irreversible covalent bond between pcASOs and the target RNA. These ASOs strongly inhibited gene expression only when the photocrosslinking rate was faster than the random walk of branch migration. In addition, pcASOs containing CNV D or CNV K targeted the RNAs with secondary structures. These results indicate the regulatory effect of photocrosslinker and photoirradiation energy using pcASOs on the gene expression level.

Psoralen inhibits malignant proliferation and induces apoptosis through triggering endoplasmic reticulum stress in human SMMC7721 hepatoma cells

BACKGROUND: Psoralen is a coumarin-like and coumarin-related benzofuran glycoside, which is a commonly used traditional Chinese medicine to treat patients with kidney and spleen-yang deficiency symptom. Psoralen has been reported to show estrogen-like activity, antioxidant activity, osteoblastic proliferation accelerating activity, antitumor effects and antibacterial activity. However, the antitumor mechanism of psoralen is not fully understood. This study aimed to investigate the therapeutic efficacy of psoralen in human hepatoma cell line SMMC7721 and the mechanism of antitumor effects. RESULTS: Psoralen inhibited proliferation of SMMC7721 in a dose- and time-dependent manner, and promoted apoptosis. Further, psoralen activated the ER stress signal pathway, including the expansion of endoplasmic reticulum, increasing the mRNA levels of GRP78, DDIT3, ATF4, XBP1, GADD34 and the protein levels of GDF15, GRP78, IRE1α, XBP-1s in a time-dependent manner. Psoralen induces cell cycle arrest at G1 phase by enhancing CyclinD1 and reducing CyclinE1 expression. Moreover, TUDC couldn't inhibit the psoralen-induced ER stress in SMMC7721 cells. CONCLUSIONS: Psoralen can inhibit the proliferation of SMMC7721 cells and induce ER stress response to induce cell apoptosis, suggesting that psoralen may represent a novel therapeutic option for the prevention and treatment hepatocellular carcinoma.