Anthocyanin Attenuates Doxorubicin-Induced Cardiomyotoxicity via Estrogen Receptor-α/β and Stabilizes HSF1 to Inhibit the IGF-IIR Apoptotic Pathway

Doxorubicin (Dox) is extensively used for chemotherapy in different types of cancer, but its use is limited to because of its cardiotoxicity. Our previous studies found that doxorubicin-induced insulin-like growth factor II receptor (IGF-IIR) accumulation causes cardiomyocytes apoptosis via down-regulation of HSF1 pathway. In these studies, we demonstrated a new mechanism through which anthocyanin protects cardiomyoblast cells against doxorubicin-induced injury. We found that anthocyanin decreased IGF-IIR expression via estrogen receptors and stabilized heat shock factor 1 (HSF1) to inhibit caspase 3 activation and apoptosis of cardiomyocytes. Therefore, the phytoestrogen from plants has been considered as another potential treatment for heart failure. It has been reported that the natural compound anthocyanin (ACN) has the ability to reduce the risk of cardiovascular disease (CVD). Here, we demonstrated that anthocyanin acts as a cardioprotective drug against doxorubicin-induced heart failure by attenuating cardiac apoptosis via estrogen receptors to stabilize HSF1 expression and down-regulated IGF-IIR-induced cardiomyocyte apoptosis.     

Cardiosulfa induces heart deformation in zebrafish through the AhR-mediated, CYP1A-independent pathway

Heart development is a complicated and elaborate biological process. To study this and similar complicated process and diseases, the discovery and use of small molecules for probing biological events is invaluable. As part of such an investigation, we have identified cardiosulfa, a small molecule that induces severely impaired heart morphology and function in zebrafish. The results of the present study show that cardiosulfa-promoted heart deformation is protected by negative regulators of the aryl hydrocarbon receptor (AhR) signaling pathway, such as the AhR antagonist CH-223191 and an AhR2-morpholino antisense oligonucleotide, zfahr2-MO. However, the toxic effect of cardiosulfa is not alleviated by zfcyp1a-MO, a morpholino antisense oligo for cytochrome P450 1A (CYP1A), which is the most well-characterized gene of the AhR pathway. Similar results were obtained for the known AhR agonist PCB126. These observations suggest that cardiosulfa causes heart deformation in zebrafish through the AhR-mediated, CYP1A-independent pathway. Our results indicate that cardiosulfa has potential as a novel type of a biological probe to investigate the AhR pathway.     

Cinnamic Acid Derivatives as Cardioprotective Agents against Oxidative and Structural Damage Induced by Doxorubicin

Doxorubicin (DOX) is a widely used anticancer drug. However, its clinical use is severely limited due to drug-induced cumulative cardiotoxicity, which leads to progressive cardiomyocyte dysfunction and heart failure. Enormous efforts have been made to identify potential strategies to alleviate DOX-induced cardiotoxicity; however, to date, no universal and highly effective therapy has been introduced. Here we reported that cinnamic acid (CA) derivatives exert a multitarget protective effect against DOX-induced cardiotoxicity. The experiments were performed on rat cardiomyocytes (H9c2) and human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) as a well-established model for cardiac toxicity assessment. CA derivatives protected cardiomyocytes by ameliorating DOX-induced oxidative stress and viability reduction. Our data indicated that they attenuated the chemotherapeutic’s toxicity by downregulating levels of caspase-3 and -7. Pre-incubation of cardiomyocytes with CA derivatives prevented DOX-induced motility inhibition in a wound-healing assay and limited cytoskeleton rearrangement. Detailed safety analyses—including hepatotoxicity, mutagenic potential, and interaction with the hERG channel—were performed for the most promising compounds. We concluded that CA derivatives show a multidirectional protective effect against DOX-induced cardiotoxicity. The results should encourage further research to elucidate the exact molecular mechanism of the compounds’ activity. The lead structure of the analyzed CA derivatives may serve as a starting point for the development of novel therapeutics to support patients undergoing DOX therapy.     

Generation of a Triadin KnockOut Syndrome Zebrafish Model

Different forms of sudden cardiac death have been described, including a recently identified form of genetic arrhythmogenic disorder, named “Triadin KnockOut Syndrome” (TKOS). TKOS is associated with recessive mutations in the TRDN gene, encoding for TRIADIN, but the pathogenic mechanism underlying the malignant phenotype has yet to be completely defined. Moreover, patients with TKOS are often refractory to conventional treatment, substantiating the need to identify new therapeutic strategies in order to prevent or treat cardiac events. The zebrafish (Danio rerio) heart is highly comparable to the human heart in terms of functions, signal pathways and ion channels, representing a good model to study cardiac disorders. In this work, we generated the first zebrafish model for trdn loss-of-function, by means of trdn morpholino injections, and characterized its phenotype. Although we did not observe any gross cardiac morphological defect between trdn loss-of-function embryos and controls, we found altered cardiac rhythm that was recovered by the administration of arrhythmic drugs. Our model will provide a suitable platform to study the effect of TRDN mutations and to perform drug screening to identify new pharmacological strategies for patients carrying TRDN mutations.     

A Reliable High-Throughput Screening Model for Antidepressant

Depression is the most frequent affective disorder and is the leading cause of disability worldwide. In order to screen antidepressants and explore molecular mechanisms, a variety of animal models were used in experiments, but there is no reliable high-throughput screening method. Zebrafish is a common model organism for mental illness such as depression. In our research, we established chronic unpredictable mild stress (CUMS) models in C57BL/6 mice and zebrafish; the similarities in behavior and pathology suggest that zebrafish can replace rodents as high-throughput screening organisms. Stress mice (ip., 1 mg/kg/d, 3 days) and zebrafish (10 mg/L, 20 min) were treated with reserpine. As a result, reserpine caused depression-like behavior in mice, which was consistent with the results of the CUMS mice model. Additionally, reserpine reduced the locomotor ability and exploratory behavior of zebrafish, which was consistent with the results of the CUMS zebrafish model. Further analysis of the metabolic differences showed that the reserpine-induced zebrafish depression model was similar to the reserpine mice model and the CUMS mice model in the tyrosine metabolism pathway. The above results showed that the reserpine-induced depression zebrafish model was similar to the CUMS model from phenotype to internal metabolic changes and can replace the CUMS model for antidepressants screening. Moreover, the results from this model were obtained in a short time, which can shorten the cycle of drug screening and achieve high-throughput screening. Therefore, we believe it is a reliable high-throughput screening model.     

An efficient route to selective bio-oxidation catalysts: an iterative approach comprising modeling, diversification, and screening, based on CYP102A1

Perillyl alcohol is the terminal hydroxylation product of the cheap and readily available terpene, limonene. It has high potential as an anti‐tumor substance, but is of limited availability. In principle, cytochrome P450 monooxygenases, such as the self‐sufficient CYP102A1, are promising catalysts for the oxidation of limonene or other inert hydrocarbons. The wild‐type enzyme converts (4R)‐limonene to four different oxidation products; however, terminal hydroxylation at the allylic C7 is not observed. Here we describe a generic strategy to engineer this widely used enzyme to hydroxylate exclusively the exposed, but chemically less reactive, primary C7 in the presence of other reactive positions. The approach presented here turns CYP102A1 into a highly selective catalyst with a shifted product spectra by successive rounds of modeling, the design of small focused libraries, and screening. In the first round a minimal CYP102A1 mutant library was rationally designed. It contained variants with improved or strongly shifted regio‐, stereo‐ and chemoselectivity, compared to wild‐type. From this library the variant with the highest perillyl alcohol ratio was fine‐tuned by two additional rounds of molecular modeling, diversification, and screening. In total only 29 variants needed to be screened to identify the triple mutant A264V/A238V/L437F that converts (4R)‐limonene to perillyl alcohol with a selectivity of 97 %. Focusing mutagenesis on a small number of relevant positions identified by computational approaches is the key for efficient screening for enzyme selectivity.     

Improved Model of Lanosterol 14α‐Demethylase by Ligand‐Supported Homology Modeling: Validation by Virtual Screening and Azole Optimization

Lanosterol 14α‐demethylase (CYP51) is an important target for antifungal drugs. An improved three‐dimensional model of CYP51 from Candida albicans (CACYP51) was constructed by ligand‐supported homology modeling and molecular dynamics simulations. The accuracy of the constructed model was evaluated by its performance in a small‐scale virtual screen. The results show that known CYP51 inhibitors were efficiently discriminated by the model, and it performed better than our previous CACYP51 model. The active site of CACYP51 was characterized by multiple copy simultaneous search (MCSS) calculations. On the basis of the MCSS results, a series of novel azoles were designed and synthesized, and they showed good in vitro antifungal activity with a broad spectrum. The MIC₈₀ value of four of these compounds against C. albicans is 0.001 μg mL^?1, indicating that they are promising leads for the discovery of novel antifungal agents.     

Efficient Neuroprotective Rescue of Sacsin-Related Disease Phenotypes in Zebrafish

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a multisystem hereditary ataxia associated with mutations in SACS, which encodes sacsin, a protein of still only partially understood function. Although mouse models of ARSACS mimic largely the disease progression seen in humans, their use in the validation of effective therapies has not yet been proposed. Recently, the teleost Danio rerio has attracted increasing attention as a vertebrate model that allows rapid and economical screening, of candidate molecules, and thus combines the advantages of whole-organism phenotypic assays and in vitro high-throughput screening assays. Through CRISPR/Cas9-based mutagenesis, we generated and characterized a zebrafish sacs-null mutant line that replicates the main features of ARSACS. The sacs-null fish showed motor impairment, hindbrain atrophy, mitochondrial dysfunction, and reactive oxygen species accumulation. As proof of principle for using these mutant fish in high-throughput screening studies, we showed that both acetyl-DL-leucine and tauroursodeoxycholic acid improved locomotor and biochemical phenotypes in sacs^−/− larvae treated with these neuroprotective agents, by mediating significant rescue of the molecular functions altered by sacsin loss. Taken together, the evidence here reported shows the zebrafish to be a valuable model organism for the identification of novel molecular mechanisms and for efficient and rapid in vivo optimization and screening of potential therapeutic compounds. These findings may pave the way for new interventions targeting the earliest phases of Purkinje cell degeneration in ARSACS.     

Characterization of CYP154F1 from Thermobifida fusca YX and Extension of Its Substrate Spectrum by Site‐Directed Mutagenesis

Previous studies on cytochrome P450 monooxygenases (CYP) from family 154 reported their substrate promiscuity and high activity. Hence, herein, the uncharacterized family member CYP154F1 is described. Screening of more than 100 organic compounds revealed that CYP154F1 preferably accepts small linear molecules with a carbon chain length of 8–10 atoms. In contrast to thoroughly characterized CYP154E1, CYP154F1 has a much narrower substrate spectrum and lower activity. A structural alignment of homology models of CYP154F1 and CYP154E1 revealed few differences in the active sites of both family members. By gradual mutagenesis of the CYP154F1 active site towards those of CYP154E1, a key residue accounting for the different activities of both enzymes was identified at position 234. Substitution of T234 for large hydrophobic amino acids led to up to tenfold higher conversion rates of small substrates, such as geraniol. Replacement of T234 by small hydrophobic amino acids, valine or alanine, resulted in mutants with extended substrate spectra. These mutants are able to convert some of the larger substrates of CYP154E1, such as (E)‐stilbene and (+)‐nootkatone.     

Expanding the Binding Envelope of CYP51 Inhibitors Targeting Trypanosoma cruzi with 4‐Aminopyridyl‐Based Sulfonamide Derivatives

Chagas disease is a chronic infection caused by the protozoan parasite Trypanosoma cruzi, manifested in progressive cardiomyopathy and/or gastrointestinal dysfunction. Therapeutic options to prevent or treat Chagas disease are limited. CYP51, the enzyme key to the biosynthesis of eukaryotic membrane sterols, is a validated drug target in both fungi and T. cruzi. Sulfonamide derivatives of 4‐aminopyridyl‐based inhibitors of T. cruzi CYP51 (TcCYP51), including the sub‐nanomolar compound 3 , have molecular structures distinct from other validated CYP51 inhibitors. They augment the biologically relevant chemical space of molecules targeting TcCYP51. In a 2.08 Å X‐ray structure, TcCYP51 is in a conformation that has been influenced by compound 3 and is distinct from the previously characterized ground‐state conformation of CYP51 drug–target complexes. That the binding site was modulated in response to an incoming inhibitor for the first time characterizes TcCYP51 as a flexible target rather than a rigid template.     

Zebrafish Larvae Behavior Models as a Tool for Drug Screenings and Pre-Clinical Trials: A Review

To discover new molecules or review the biological activity and toxicity of therapeutic substances, drug development, and research relies on robust biological systems to obtain reliable results. Phenotype-based screenings can transpose the organism’s compensatory pathways by adopting multi-target strategies for treating complex diseases, and zebrafish emerged as an important model for biomedical research and drug screenings. Zebrafish’s clear correlation between neuro-anatomical and physiological features and behavior is very similar to that verified in mammals, enabling the construction of reliable and relevant experimental models for neurological disorders research. Zebrafish presents highly conserved physiological pathways that are found in higher vertebrates, including mammals, along with a robust behavioral repertoire. Moreover, it is very sensitive to pharmacological/environmental manipulations, and these behavioral phenotypes are detected in both larvae and adults. These advantages align with the 3Rs concept and qualify the zebrafish as a powerful tool for drug screenings and pre-clinical trials. This review highlights important behavioral domains studied in zebrafish larvae and their neurotransmitter systems and summarizes currently used techniques to evaluate and quantify zebrafish larvae behavior in laboratory studies.     

斑马鱼模型在化妆品研究中的应用

化妆品已成为人们生活中的必需品,安全性和功效性评价是化妆品开发过程中非常重要的一个环节。斑马鱼具有传代时间短、饲养方便、价格低廉以及便于大规模筛选等优势,是一种有效、可靠、高通量的模式生物。目前,对斑马鱼致死和致畸的统计结果可用于化妆品原料的安全性评价和防晒剂抗紫外线的功效评价,其身体表面黑色素数量的变化可用于去斑美白原料的功效评价,均呈现出良好的发展优势和应用前景。     

Synthesis of New AIEE-Active Chalcones for Imaging of Mitochondria in Living Cells and Zebrafish In Vivo

Fluorophores with aggregation-induced emission enhancement (AIEE) properties have attracted increasing interest in recent years. On the basis of our previous research, we successfully designed and synthesized eleven chalcones. Through an optical performance experiment, we confirmed that compounds 1–6 had obvious AIEE properties. As these AIEE molecules had excellent fluorescence properties and a large Stokes shift, we studied their application in living cell imaging, and the results showed that these compounds had low cytotoxicity and good biocompatibility at the experimental concentrations. More importantly, they could specifically label mitochondria. Subsequently, we selected zebrafish as experimental animals to explore the possibilities of these compounds in animal imaging. The fluorescence imaging of zebrafish showed that these AIEE molecules can enter the embryo and can be targeted to aggregate in the digestive tract, which provides a strong foundation for their practical application in the field of biological imaging. Compared with traditional fluorophores, these AIEE molecules have the advantages of possessing a small molecular weight and high flexibility. Therefore, they have excellent application prospects in the field of biological imaging. In addition, the findings of this study have very positive practical significance for the discovery of more AIEE molecules.     

Investigation of the Antiremodeling Effects of Losartan,Mirabegron and Their Combination on the Development of Doxorubicin-Induced Chronic Cardiotoxicity in a Rat Model

Despite the effectiveness of doxorubicin (DOXO) as a chemotherapeutic agent, dose-dependent development of chronic cardiotoxicity limits its application. The angiotensin-II receptor blocker losartan is commonly used to treat cardiac remodeling of various etiologies. The beta-3 adrenergic receptor agonist mirabegron was reported to improve chronic heart failure. Here we investigated the effects of losartan, mirabegron and their combination on the development of DOXO-induced chronic cardiotoxicity. Male Wistar rats were divided into five groups: (i) control; (ii) DOXO-only; (iii) losartan-treated DOXO; (iv) mirabegron-treated DOXO; (v) losartan plus mirabegron-treated DOXO groups. The treatments started 5 weeks after DOXO administration. At week 8, echocardiography was performed. At week 9, left ventricles were prepared for histology, qRT-PCR, and Western blot measurements. Losartan improved diastolic but not systolic dysfunction and ameliorated SERCA2a repression in our DOXO-induced cardiotoxicity model. The DOXO-induced overexpression of Il1 and Il6 was markedly decreased by losartan and mirabegron. Mirabegron and the combination treatment improved systolic and diastolic dysfunction and significantly decreased overexpression of Smad2 and Smad3 in our DOXO-induced cardiotoxicity model. Only mirabegron reduced DOXO-induced cardiac fibrosis significantly. Mirabegron and its combination with losartan seem to be promising therapeutic tools against DOXO-induced chronic cardiotoxicity.     

Helicoverpa zea CYP6B8 and CYP321A1: different molecular solutions to the problem of metabolizing plant toxins and insecticides

Under continual exposure to naturally occurring plant toxins and synthetic insecticides, insects have evolved cytochrome P450 monooxygenases (P450s) capable of metabolizing a wide range of structurally different compounds. Two such P450s, CYP6B8 and CYP321A1, expressed in Helicoverpa zea (a lepidopteran) in response to plant allelochemicals and plant signaling molecules metabolize these compounds with varying efficiencies. While sequence alignments of these proteins indicate highly divergent substrate recognition sites (SRSs), homology models developed for them indicate that the two active site cavities have essentially the same volume with distinct shapes dictated by side-chain differences in SRS1 and SRS5. CYP6B8 has a narrower active site cavity extending from substrate access channel pw2a with a very narrow access to the ferryl oxygen atom. This predicted shape suggests that bulkier molecules bind further from the ferryl oxygen at positions that are not as effectively metabolized. In contrast, CYP321A1 is predicted to have a more spacious cavity allowing larger molecules to access the heme-bound oxygen. The metabolic profiles for several plant toxins (xanthotoxin, angelicin) and insecticides (cypermethrin, aldrin and diazinon) correlate well with these predictive models. The absence of Thr in the I helix of CYP321A1 and hydroxyl groups on many of its substrates suggests that this insect P450 mediates oxygen activation by a mechanism different from that employed by CYP107A1 and CYP158A1, which are two bacterial P450s also lacking Thr in their I helix, and most other P450s that contain Thr in their I helix.     

CYP109E1 from Bacillus megaterium Acts as a 24- and 25-Hydroxylase for Cholesterol

In this study, the ability of CYP109E1 from Bacillus megaterium DSM319 to metabolize cholesterol was investigated. This steroid was identified as a new substrate to be converted by CYP109E1 with adrenodoxin and adrenodoxin reductase as redox partners in vitro. The biotransformation was successfully reproduced in vivo by using Bacillus megaterium cells that overexpressed CYP109E1. To enhance the production of cholesterol derivatives, an Escherichia coli based whole-cell system that harbored CYP109E1 was established. This novel system showed a 3.3-fold higher activity than that of the B. megaterium system, yielding about 45 mg L⁻¹ of these products. Finally, the reaction products were isolated and identified to be the highly important cholesterol derivatives 24(S)- and 25-hydroxycholesterol.     

Gyrase ATPase Domain as an Antitubercular Drug Discovery Platform: Structure‐Based Design and Lead Optimization of Nitrothiazolyl Carboxamide Analogues

In this study, we explored the pharmaceutically underexploited mycobacterial gyrase ATPase (GyrB) domain as a template for a structure‐based virtual screening of our in‐house (BITS Pilani) compound collection to discover new inhibitors targeting Mycobacterium tuberculosis (M.tb.) The hit identified was further customized by using a combination of molecular docking and medicinal chemistry strategies to obtain an optimized analogue displaying considerable in vitro enzyme efficacy and bactericidal properties against the M.tb. H₃₇Rv strain. The binding affinity of the ligand toward the GyrB domain was reascertained by differential scanning fluorimetry experiments. Further evaluation of the hERG toxicity (a major limitation among the previously reported N‐linked aminopiperidine analogues) indicated these molecules to be completely devoid of cardiotoxicity, a significant achievement within this class.