Temperature-robust and ratiometric G-quadruplex proximate DNAzyme assay for robustly monitoring of uranium pollution and its microbial biosorbents screening.

Uranium pollution in environment and food chain is a serious threat to public security and human health. Herein, we proposed a temperature-robust, ratiometric, and label-free bioassay based on G-quadruplex proximate DNAzyme (G4DNAzyme), accommodating us to precisely monitor uranium pollution and biosorption. The proximity of split G-quadruplex probes was proposed to sense UO22+-activated DNAzyme activity, thus eliminating the use of chemically labeled nucleic acid probes. And the simultaneous monitoring of G-quadruplex and double-stranded structures of DNAzyme probes contributed to a ratiometric and robust detection of UO22+. Particularly, the separation of enzymatic digestion and fluorescence monitoring endued a robust and highly responsive detection of UO22+ upon the temperature of enzymatic digestion process ranged from 18° to 41 °C. Consequently, G4DNAzyme assay allowed a robust, label-free and ratiometric quantification of uranium. We demonstrated the feasibility of G4DNAzyme assay for estimating uranium pollution in water and aquatic product samples. Ultimately, G4DNAzyme assay was adopted to serve as the platform to screen bacterial species and conditions for uranium biosorption, promising its roles in uranium associated biosafety control.

Homotherapy for heteropathy active components and mechanisms of Qiang-Huo-Sheng-Shi decoction for treatment of rheumatoid arthritis and osteoarthritis.

Qiang-Huo-Sheng-Shi decoction (QHSSD), a classic traditional Chinese herbal formula, which has been reported to be effective in rheumatoid arthritis (RA) and osteoarthritis (OA). However, the concurrent targeting mechanism of how the aforementioned formula is valid in the two distinct diseases OA and RA, which represents the homotherapy-for-heteropathy principle in traditional Chinese medicine (TCM), have not yet been clarified. In the present study, network pharmacology was adopted to analyze the potential molecular mechanism, and therapeutic effective components of QHSSD on both OA and RA. A total of 153 active ingredients in QHSSD were identified, 142 of which associated with 59 potential targets for the two diseases were identified. By constructing the protein-protein interaction network and the compound-target-disease network, 72 compounds and 10 proteins were obtained as the hub targets of QHSSD against OA and RA. The hub genes of ESR1, PTGS2, PPARG, IL1B, TNF, MMP2, IL6, CYP3A4, MAPK8, and ALB were mainly involved in osteoclast differentiation, the NF-κB and TNF signaling pathways. Moreover, molecular docking results showed that the screened active compounds had a high affinity for the hub genes. This study provides new insight into the molecular mechanisms behind how QHSSD presents homotherapy-for-heteropathy therapeutic efficacy in both OA and RA. For the first time, a two-disease model was linked with a TCM formula using network pharmacology to identify the key active components and understand the common mechanisms of its multi-pathway regulation. This study will inspire more innovative and important studies on the modern research of TCM formulas.

Xie2-64, a novel CB2 receptor inverse agonist, reduces cocaine abuse-related behaviors in rodents.

Cocaine abuse remains a public health threat around the world. There are no pharmacological treatments approved for cocaine use disorder. Cannabis has received growing attention as a treatment for many conditions, including addiction. Most cannabis-based medication development has focused on cannabinoid CB1 receptor (CB1R) antagonists (and also inverse agonists) such as rimonabant, but clinical trials with rimonabant have failed due to its significant side-effects. Here we sought to determine whether a novel and selective CB2R inverse agonist, Xie2-64, has similar therapeutic potential for cocaine use disorder. Computational modeling indicated that Xie2-64 binds to CB2R in a way similar to SR144528, another well-characterized but less selective CB2R antagonist/inverse agonist, suggesting that Xie2-64 may also have CB2R antagonist profiles. Unexpectedly, systemic administration of Xie2-64 or SR144528 dose-dependently inhibited intravenous cocaine self-administration and shifted cocaine dose-response curves downward in rats and wild-type, but not in CB2R-knockout, mice. Xie2-64 also dose-dependently attenuated cocaine-enhanced brain-stimulation reward maintained by optical stimulation of ventral tegmental area dopamine (DA) neurons in DAT-Cre mice, while Xie2-64 or SR144528 alone inhibited optical brain-stimulation reward. In vivo microdialysis revealed that systemic or local administration of Xie2-64 into the nucleus accumbens reduced extracellular dopamine levels in a dose-dependent manner in rats. Together, these results suggest that Xie2-64 has significant anti-cocaine reward effects likely through a dopamine-dependent mechanism, and therefore, deserves further study as a new pharmacotherapy for cocaine use disorder.

Effect of Black Tea and Green Tea on the Radial Pulse Spectrum in Healthy Humans.

The consumption of black tea and green tea has been shown to be beneficial for cardiovascular health. Because the chemical composition of the two teas varies widely, the purpose of the study was to investigate whether the consumption of green tea and black tea had different effects on the arterial system. Thirty-three healthy subjects received a single dose of green tea (dose = 0.05 g/kg) and black tea (dose = 0.05 g/kg) in different weeks. Radial blood pressure and radial pulse pressure were measured before and after drinking tea. The harmonic analysis was performed on radial pressure waves, and harmonics (Cns) were recorded. The results showed that both black tea and green tea consumption significantly increased the C1, C2, C6, C7, C8, C9, and C10 of the radial pressure wave. Furthermore, the results confirmed that the consumption effect of green tea on C6-C10 increase is greater than that of black tea. This report also found a subtle difference that consumption of green tea increased C4, whereas consumption of black tea increased C3. We concluded that black tea and green tea have similar patterns in higher harmonics, but with varying degrees.

Development and Testing of Druglike Screening Libraries.

Although significant advances in experimental high throughput screening (HTS) have been made for drug lead identification, in silico virtual screening (VS) is indispensable owing to its unique advantage over experimental HTS, target-focused, cheap, and efficient, albeit its disadvantage of producing false positive hits. For both experimental HTS and VS, the quality of screening libraries is crucial and determines the outcome of those studies. In this paper, we first reviewed the recent progress on screening library construction. We realized the urgent need for compiling high-quality screening libraries in drug discovery. Then we compiled a set of screening libraries from about 20 million druglike ZINC molecules by running fingerprint-based similarity searches against known drug molecules. Lastly, the screening libraries were objectively evaluated using 5847 external actives covering more than 2000 drug targets. The result of the assessment is very encouraging. For example, with the Tanimoto coefficient being set to 0.75, 36% of external actives were retrieved and the enrichment factor was 13. Additionally, drug target family specific screening libraries were also constructed and evaluated. The druglike screening libraries are available for download from https://mulan.pharmacy.pitt.edu .

Indirubin ameliorates imiquimod-induced psoriasis-like skin lesions in mice by inhibiting inflammatory responses mediated by IL-17A-producing γδ T cells.

OBJECTIVES: Indirubin (IR) is a bisindole compound extracted from the leaves of Chinese herb Indigo Naturalis. Indigo Naturalis has been widely used in traditional Chinese medicine to treat inflammatory and autoimmune diseases. Psoriasis is a chronic immune-mediated inflammatory skin disease in which γδ T cells play an important role. This study aims to determine the immunoregulatory effects and the underlying mechanisms of Indirubin in psoriasis-related inflammatory responses. METHODS: BALB/c mice with imiquimod (IMQ)-induced psoriasis-like dermatitis were treated with saline (Model), 1 mg/kg methotrexate (MTX) that serves as a positive control, or 12.5, 25 and 50 mg/kg Indirubin(IR) intragastrically. Keratinocytes proliferation, inflammatory cells infiltration, the expression of inflammatory cytokines and Jak/Stat pathway-related proteins in the skin lesion were examined. The abundance of γδ T cells in lymph nodes and spleen was determined by flow cytometry. The IL-17 expression and secretion, and the activation of Jak3/Stat3 pathways in in vitro cultured γδ T cell were tested. RESULTS: Indirubin ameliorated keratinocyte proliferation, reduced the infiltration of CD3+ T cells, IL-17 A-producing γδ T cells, and CD11b+ neutrophils, inhibited the mRNA expression of Il1, Il6, Il23, Il17a and Il22, and the protein expression of Jak/Stat pathway-related molecules in the skin lesion. Indirubin also reduced the abundance of γδ T cell and CCR6+ γδ T cells (the major IL-17 A producer) in spleen and lymph nodes. In cultured γδ T cells, Indirubin inhibited the mRNA expression of Il17a and Ifng, and the secretion of IL-17 A, while suppressed the activation of Jak3/Stat3 pathways. CONCLUSION: Indirubin alleviates IMQ-induced psoriasis-like dermatitis mainly through reducing the inflammatory responses mediated by IL-17 A-producing γδ T cells involving Jak3/Stat3 activation. Our results highlighted the novel mechanisms by which Indirubin ameliorates psoriasis-related inflammatory responses, supporting its therapeutic potential.

Cardiovascular Disease Chemogenomics Knowledgebase-guided Target Identification and Drug Synergy Mechanism Study of an Herbal Formula.

Combination therapy is a popular treatment for various diseases in the clinic. Among the successful cases, Traditional Chinese Medicinal (TCM) formulae can achieve synergistic effects in therapeutics and antagonistic effects in toxicity. However, characterizing the underlying molecular synergisms for the combination of drugs remains a challenging task due to high experimental expenses and complication of multicomponent herbal medicines. To understand the rationale of combination therapy, we investigated Sini Decoction, a well-known TCM consisting of three herbs, as a model. We applied our established diseases-specific chemogenomics databases and our systems pharmacology approach TargetHunter to explore synergistic mechanisms of Sini Decoction in the treatment of cardiovascular diseases. (1) We constructed a cardiovascular diseases-specific chemogenomics database, including drugs, target proteins, chemicals, and associated pathways. (2) Using our implemented chemoinformatics tools, we mapped out the interaction networks between active ingredients of Sini Decoction and their targets. (3) We also in silico predicted and experimentally confirmed that the side effects can be alleviated by the combination of the components. Overall, our results demonstrated that our cardiovascular disease-specific database was successfully applied for systems pharmacology analysis of a complicated herbal formula in predicting molecular synergetic mechanisms, and led to better understanding of a combinational therapy.

Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery.

Cannabinoid receptor 2 (CB2), a G protein-coupled receptor (GPCR), is a promising target for the treatment of neuropathic pain, osteoporosis, immune system, cancer, and drug abuse. The lack of an experimental three-dimensional CB2 structure has hindered not only the development of studies of conformational differences between the inactive and active CB2 but also the rational discovery of novel functional compounds targeting CB2. In this work, we constructed models of both inactive and active CB2 by homology modeling. Then we conducted two comparative 100 ns molecular dynamics (MD) simulations on the two systems-the active CB2 bound with both the agonist and G protein and the inactive CB2 bound with inverse agonist-to analyze the conformational difference of CB2 proteins and the key residues involved in molecular recognition. Our results showed that the inactive CB2 and the inverse agonist remained stable during the MD simulation. However, during the MD simulations, we observed dynamical details about the breakdown of the "ionic lock" between R131(3.50) and D240(6.30) as well as the outward/inward movements of transmembrane domains of the active CB2 that bind with G proteins and agonist (TM5, TM6, and TM7). All of these results are congruent with the experimental data and recent reports. Moreover, our results indicate that W258(6.48) in TM6 and residues in TM4 (V164(4.56)-L169(4.61)) contribute greatly to the binding of the agonist on the basis of the binding energy decomposition, while residues S180-F183 in extracellular loop 2 (ECL2) may be of importance in recognition of the inverse agonist. Furthermore, pharmacophore modeling and virtual screening were carried out for the inactive and active CB2 models in parallel. Among all 10 hits, two compounds exhibited novel scaffolds and can be used as novel chemical probes for future studies of CB2. Importantly, our studies show that the hits obtained from the inactive CB2 model mainly act as inverse agonist(s) or neutral antagonist(s) at low concentration. Moreover, the hit from the active CB2 model also behaves as a neutral antagonist at low concentration. Our studies provide new insight leading to a better understanding of the structural and conformational differences between two states of CB2 and illuminate the effects of structure on virtual screening and drug design.

Structural insight into tetrameric hTRPV1 from homology modeling, molecular docking, molecular dynamics simulation, virtual screening, and bioassay validations.

The transient receptor potential vanilloid type 1 (TRPV1) is a heat-activated cation channel protein, which contributes to inflammation, acute and persistent pain. Antagonists of human TRPV1 (hTRPV1) represent a novel therapeutic approach for the treatment of pain. Developing various antagonists of hTRPV1, however, has been hindered by the unavailability of a 3D structure of hTRPV1. Recently, the 3D structures of rat TRPV1 (rTRPV1) in the presence and absence of ligand have been reported as determined by cryo-EM. rTRPV1 shares 85.7% sequence identity with hTRPV1. In the present work, we constructed and reported the 3D homology tetramer model of hTRPV1 based on the cryo-EM structures of rTRPV1. Molecular dynamics (MD) simulations, energy minimizations, and prescreen were applied to select and validate the best model of hTRPV1. The predicted binding pocket of hTRPV1 consists of two adjacent monomers subunits, which were congruent with the experimental rTRPV1 data and the cyro-EM structures of rTRPV1. The detailed interactions between hTRPV1 and its antagonists or agonists were characterized by molecular docking, which helped us to identify the important residues. Conformational changes of hTRPV1 upon antagonist/agonist binding were also explored by MD simulation. The different movements of compounds led to the different conformational changes of monomers in hTRPV1, indicating that TRPV1 works in a concerted way, resembling some other channel proteins such as aquaporins. We observed that the selective filter was open when hTRPV1 bound with an agonist during MD simulation. For the lower gate of hTRPV1, we observed large similarities between hTRPV1 bound with antagonist and with agonist. A five-point pharmacophore model based on several antagonists was established, and the structural model was used to screen in silico for new antagonists for hTRPV1. By using the 3D TRPV1 structural model above, the pilot in silico screening has begun to yield promising hits with activity as hTRPV1 antagonists, several of which showed substantial potency.

Effects of Low-Protein Diets Supplemented with Ketoacid on Expression of TGF-ß and Its Receptors in Diabetic Rats.

TGF-ß 1 has been recognized as a key mediator in DN. This study aimed to observe the effects of low-protein diets supplemented with ketoacid on mRNA and protein expression of TGF-ß and TßRI and t TßRII receptors in the renal tissue of diabetic rats. A diabetes model was established in 72 male SD rats. They were then equally randomized to three groups: NPD group, LPD group, and LPD + KA group. Additional 24 male SD rats receiving normal protein diets were used as the control. Eight rats from each group were sacrificed at weeks 4, 8, and 12 after treatment, from which SCr, BUN, serum albumin, and 24 h urinary protein excretion were collected. The expressions of TGF-ß 1, TßRI, and TßRII in LPD and LPD + KA groups were significantly lower than those in NPD group and lower in LPD + KA group than those in LPD group. Low-protein diets supplemented with ketoacid have been demonstrated to provide a protective effect on the renal function as represented by reduced SCr, BUN, and urinary protein excretion, probably through downregulating the gene expression of TGF-ß 1 and its receptors in LPD + KA group.

Preparation, characterization, and pharmacodynamics of thermosensitive liposomes containing docetaxel.

A novel thermosensitive liposome (TL) containing docetaxel (DTX) was designed to enhance DTX-targeted delivery and antitumor effect. TL loading DTX (DTX-TL) were prepared by thin film hydration. The mean particle size of the liposomes was about 100 nm, and the drug entrapment efficiency was more than 95%. The phase transition temperature of liposomes was about 42 °C. In vitro drug release showed that drug released at 37 °C was obviously less than that at 42 °C. For in vivo experiments, the human breast tumor model was established by subcutaneous xenotransplantation on nude mice; liposomes and injection containing DTX were injected i.v. in nude mice, followed by exposure of the tumors to hyperthermia (HT) for 30 min after administration. The tumor inhibition ratio of DTX-TL group was significantly higher than the normal injection group. Combining TL with HT enhanced the delivery of DTX and thereby its antitumor effects. The liposomes reported in this paper could potentially produce viable clinical strategies for improved targeting and delivery of DTX for the treatment of breast cancer.

Comparative normal/failing rat myocardium cell membrane chromatographic analysis system for screening specific components that counteract doxorubicin-induced heart failure from Acontium carmichaeli.

Cell membrane chromatography (CMC) derived from pathological tissues is ideal for screening specific components acting on specific diseases from complex medicines owing to the maximum simulation of in vivo drug-receptor interactions. However, there are no pathological tissue-derived CMC models that have ever been developed, as well as no visualized affinity comparison of potential active components between normal and pathological CMC columns. In this study, a novel comparative normal/failing rat myocardium CMC analysis system based on online column selection and comprehensive two-dimensional (2D) chromatography/monolithic column/time-of-flight mass spectrometry was developed for parallel comparison of the chromatographic behaviors on both normal and pathological CMC columns, as well as rapid screening of the specific therapeutic agents that counteract doxorubicin (DOX)-induced heart failure from Acontium carmichaeli (Fuzi). In total, 16 potential active alkaloid components with similar structures in Fuzi were retained on both normal and failing myocardium CMC models. Most of them had obvious decreases of affinities on failing myocardium CMC compared with normal CMC model except for four components, talatizamine (TALA), 14-acetyl-TALA, hetisine, and 14-benzoylneoline. One compound TALA with the highest affinity was isolated for further in vitro pharmacodynamic validation and target identification to validate the screen results. Voltage-dependent K(+) channel was confirmed as a binding target of TALA and 14-acetyl-TALA with high affinities. The online high throughput comparative CMC analysis method is suitable for screening specific active components from herbal medicines by increasing the specificity of screened results and can also be applied to other biological chromatography models.

Target-selective phototherapy using a ligand-based photosensitizer for type 2 cannabinoid receptor.

Phototherapy is a powerful, noninvasive approach for cancer treatment, with several agents currently in clinical use. Despite the progress and promise, most current phototherapy agents have serious side effects as they can lead to damage to healthy tissue, even when the photosensitizers are fused to targeting molecules due to nonspecific light activation of the unbound photosensitizer. To overcome these limitations, we developed a phototherapy agent that combines a functional ligand and a near infrared phthalocyanine dye. Our target is type 2 cannabinoid receptor (CB2R), considered an attractive therapeutic target for phototherapy given it is overexpressed by many types of cancers that are located at a surface or can be reached by an endoscope. We show that our CB2R-targeted phototherapy agent, IR700DX-mbc94, is specific for CB2R and effective only when bound to the target receptor. Overall, this opens up the opportunity for development of an alternative treatment option for CB2R-positive cancers.

Lifespan extension by n-butanol extract from seed of Platycladus orientalis in Caenorhabditis elegans.

AIM OF THE STUDY: As a traditional Chinese medicine, seed of Platycladus orientalis(Linnaeus) Franco has been extensively used as a tonic and sedative remedy. The present study was conducted to investigate whether lifespan was extended and the mechanisms of n-butanol extract from seed of Platycladus orientalis (BSPO) in Caenorhabditis elegans. The findings could provide the pharmacological basis for a treatment in traditional medicine. MATERIALS AND METHODS: Lifespan extension by BSPO was evaluated under normal culture conditions and in a stress test. A possible mechanism of the anti-aging effect of BSPO, a change in the stress-resistance of related proteins, was also investigated in C. elegans. RESULTS: It has been shown that BSPO could significantly extend lifespan of C. elegans in a concentration dependent manner under normal culture conditions and stress. Further studies demonstrated that BSPO treatment significantly decreased reactive oxygen species (ROS) accumulation, up-regulated resistance to stress of related proteins, including glutathione S-transferase-4 (GST-4) and heat shock protein-16.2 (HSP-16.2), and reduced the amount of lipofuscin in transgenic C. elegans. CONCLUSION: These results indicated that BSPO extended the lifespan, which could be attributed to its direct ROS scavenging activity, reducing the amount of lipofuscin and increasing the expression of gens associated with resistance to stress. These obtained data provided valuable support for traditional clinical practice to extend lifespan and to provide tonic remedy.

Identification and characterization of human apurinic/apyrimidinic endonuclease-1 inhibitors.

The repair of abasic sites that arise in DNA from hydrolytic depurination/depyrimidination of the nitrogenous bases from the sugar-phosphate backbone and the action of DNA glycosylases on deaminated, oxidized, and alkylated bases are critical to cell survival. Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1; aka APE1/ref-1) is responsible for the initial removal of abasic lesions as part of the base excision repair pathway. Deletion of APE-1 activity is embryonic lethal in animals and is lethal in cells. Potential inhibitors of the repair function of APE-1 were identified based upon molecular modeling of the crystal structure of the APE-1 protein. We describe the characterization of several unique nanomolar inhibitors using two complementary biochemical screens. The most active molecules all contain a 2-methyl-4-amino-6,7-dioxolo-quinoline structure that is predicted from the modeling to anchor the compounds in the endonuclease site of the protein. The mechanism of action of the selected compounds was probed by fluorescence and competition studies, which indicate, in a specific case, direct interaction between the inhibitor and the active site of the protein. It is demonstrated that the inhibitors induce time-dependent increases in the accumulation of abasic sites in cells at levels that correlate with their potency to inhibit APE-1 endonuclease excision. The inhibitor molecules also potentiate by 5-fold the toxicity of a DNA methylating agent that creates abasic sites. The molecules represent a new class of APE-1 inhibitors that can be used to probe the biology of this critical enzyme and to sensitize resistant tumor cells to the cytotoxicity of clinically used DNA damaging anticancer drugs.

Alkylamides from Echinacea are a new class of cannabinomimetics. Cannabinoid type 2 receptor-dependent and -independent immunomodulatory effects.

Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor alpha mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB2) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563-569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB2 receptor more strongly than the endogenous cannabinoids. The Ki values of A1 and A2 (CB2 approximately 60 nM; CB1 >1500 nM) were determined by displacement of the synthetic high affinity cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB2, directed by H-bonding and pi-pi interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2+ in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist SR144528. At 50 nM, A1, A2, and the endogenous cannabinoid anandamide (CB2 Ki >200 nM) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB2-dependent manner. A1, A2, anandamide, the CB2 antagonist SR144528 (Ki <10 nM), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor alpha, IL-1beta, and IL-12p70 expression (5-500 nM) in a CB2-independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB2 binding.