Frontiers in Chemistry

The lack of nuclear estrogen receptor (ER and ER{beta}) bindings of 10{beta}-hydroxyestra-1,4-diene-3,17-dione (HEDD) and structurally related steroidal para-quinols have been shown by an extensive series of multidisciplinary investigational evidence including specific receptor binding studies. In support of the latter, the absence of estrogen-derived para-quinols in vivo uterotrophic effects has also been well documented. Via in silico docking, a recent publication by Canario et al. (2022) reported a robust binding of HEDD (Figure 1B) to ER. The authors claimed a strong binding of HEDD -- as strong as that of its natural ligand, 17{beta}-estradiol (E2), the main human estrogen. However, an examination of the virtual binding pocket revealed that at least one residue near the critical ligand-binding site of their reported HEDD-ER complex was labelled as "unknown" indicating thereby alteration of the receptors published structure (Tannenbaum et al, 1998; Bafna et al., 2020) to fit the ligand. Based on these arguments, the contradictory result by Canario et al. (2022) on HEDDs binding to ER should be dismissed. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/501604v2_fig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@488472org.highwire.dtl.DTLVardef@ef7642org.highwire.dtl.DTLVardef@13d1ff4org.highwire.dtl.DTLVardef@1fce259_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO (A) Schematic illustration of CNS-selective reductive bioactivation of bioprecursor prodrugs shown in panel B to the corresponding estrogen (E2, E2 or E1). (B) Chemical structures of bioprecursor prodrugs of estrogens: 10,17-dihydroxyestra-1,4-dien-3-one (DHED) for E2; 10,17-dihydroxyestra-1,4-dien-3-one (DHED) for E2, and 10-hydroxyestra-1,4-dien-3,17-dione (HEDD) for E1 (Prokai-Tatrai and Prokai, 2018). C_FIG

Ipomoeassin F (Ipom-F) is a natural compound with embedded carbohydrates that exhibits a potent cytotoxic effect on triple-negative breast cancer (TNBC) cells. The mechanism behind this selective potency remains unclear. To elucidate this mechanism, we analyzed the proteome profiles of the TNBC MDA-MB-231 cells after exposure to Ipom-F at different time points and increasing doses using a quantitative proteomic method. Our proteomic data demonstrate that the major effect of Ipom-F on MDA-MB-231 cells is the inhibition of membrane and secreted protein expression. Our proteomic data are consistent with the recently uncovered molecular mechanism of action of Ipom-F, which binds to Sec61- and inhibits the co-translational import of proteins into the endoplasmic reticulum. We have defined a subset of membrane and secreted proteins particularly sensitive to Ipom-F. Analysis of the expression of these Ipom-F-sensitive proteins in cancer cell lines and breast cancer tissues demonstrates that some of these proteins are upregulated in TNBC cells. Thus, it is likely that TNBC cells may have adapted to the elevated levels of some proteins identified as sensitive to Ipom-F in this study; inhibition of the expression of these proteins leads to a crisis in proliferation and/or survival for the cells.

Nature-derived bioactive compounds have emerged as promising candidates for the prevention and treatment of diverse chronic illnesses, including neurodegenerative diseases. However, the exact molecular mechanisms underlying their neuroprotective effects remain unclear. Most studies focus solely on the antioxidant activities of natural products which translate to poor outcome in clinical trials. Current therapies against neurodegeneration only provide symptomatic relief thereby underscoring the need for novel strategies to combat disease onset and progression. We have employed an environmental toxin-induced Drosophila Parkinsons disease (PD) model as an inexpensive in vivo screening platform to explore neuroprotective potential of selected dietary flavonoids. We have identified a specific group of flavonoids known as flavones displaying protection against paraquat (PQ)-induced neurodegenerative phenotypes, involving reduced survival, mobility defects and enhanced oxidative stress. Interestingly, the other groups of investigated flavonoids, namely, the flavonones and flavonols failed to provide protection indicating a requirement of specific structural features that confer protection against PQ-mediated neurotoxicity in Drosophila. Based on our screen, the neuroprotective flavones lack a functional group substitution at the C3 and contain ,{beta}-unsaturated carbonyl group. Furthermore, flavones-mediated neuroprotection is not solely dependent on antioxidant properties but also involves regulation of neuroinflammatory responses. Our data identify specific structural features of selected flavonoids that provide neuroprotection against environmental toxin-induced PD pathogenesis that can be explored for novel therapeutic interventions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/494711v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@130933aorg.highwire.dtl.DTLVardef@11e959org.highwire.dtl.DTLVardef@1eef5fforg.highwire.dtl.DTLVardef@1585a2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Phosphatase of Regenerating Liver-3 (PRL-3) is associated with cancer progression and metastasis in various solid tumors and leukemias. The mechanisms that drive PRL-3s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. In particular, small molecules do not exhibit binding specificity for PRL-3 over highly homologous family members PRL-1 and PRL-2, and antibodies directed against PRL-3 are limited by assay type. We have begun to address these issues by developing alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3 with a KD of 30-300 nM and no activity towards PRL-1 and PRL-2. Hydrogen deuterium exchange mass spectrometry (HDX-MS) and co-immunoprecipitation with a known PRL-3 substrate showed the nanobodies bind PRL-3 outside of the active site, meaning they can be used to study PRL-3 interaction with binding partners. The nanobodies were also specific to PRL-3 over other PRLs in immunoprecipitation and immunofluorescence experiments in human cancer cells that overexpressed the PRL family. We found that N-terminal tags on PRL-3, such as GFP and FLAG, changed PRL-3 localization compared to untagged protein, indicating that the nanobodies may provide new insights into PRL-3 trafficking and function. The anti-PRL-3 nanobodies represent an important expansion of the research tools available to study PRL-3 function and can be used to define the role of PRL-3 in cancer progression.

Natural products are a time-tested source of medicinal compounds, and there is huge interest in discovery of new drugs from plant sources. However, in many cases the effects are not as well understood, strong, or selective, as would be hoped. An example of this is madecassic acid (MA), which has promising activity against liver cancer, activity which can be improved through chemical modifications, although a molecular understanding of its mechanism of action is unknown. In this report we have used chemical proteomics to identify the proteins with which madecassic interacts in liver cancer cells, and used RNAseq to support those findings, as well as showing more precisely how chemical modifications can focus and amplify MA activity. Our results show that madecassic acid interacts with a number of proteins relating to metabolism, nucleic acid processing, and protein folding, which have been previously identified as linked to liver cancer. This provides a route from phenotypic- to target based-drug discovery and develop new potential treatments for a globally challenging disease.

The adamantane derived diaza-crown ether ZG613 was assessed as a potential breast cancer cells and breast epithelial to mesenchymal transition (EMT)-model cells targeting agent. We postulated that ZG613 activity relies on its plasma/mitochondria membrane disruption ability based on adamantane hydrophobicity and/or crown ether related ionophoric properties. We performed molecular dynamics (MD) simulations and next generation sequencing, followed by in vitro study of cell death, membrane perturbations and ionophoric ability, as well as in vivo study of effects on the tumour growth. MD simulation and RNA sequencing pointed toward physical disruption of plasma membrane by ZG613, corroborated by measured increase in membrane permeability leading to cell death. Measurements of ion fluxes confirmed ZG613 inability to transport Na+ and K+, as predicted by MD simulation. EMT-model cells exhibit changes in mitochondrial morphology and ATP levels, successfully targeted by ZG613. ZG613 caused mild retardation of tumour growth in vivo. In conclusion, ZG613 kills breast cancer cells and breast EMT-model cells by physical disruption of plasma membrane and impairments of mitochondrial functions. Breast EMT cells represent good potential targets within the breast tumour, due to their plasma membrane and mitochondrial instability.

Management of gluten intolerance is currently possible only by consumption of gluten free diet(GFD) for a lifetime. The scientific community has been searching for alternatives to GFD, like inclusion of natural proteases with meals or pre-treatment of gluten containing foods with glutenases. Actinidin from kiwifruit has shown considerable promise in digesting immunogenic gliadin peptides as compared to other plant derived cysteine proteases. Through this article, we have attempted to understand the structural basis for elevated protease action of actinidin against gliadin peptides by using an in silico approach. Docking experiments reveal key differences between the binding of gliadin peptide to actinidin and papain, which may be responsible for their differential digestive action. Sequence comparison of different plant cysteine proteases highlights amino acid residues surrounding the active site pocket of actinidin that are unique to this molecule and hence likely to contribute to its digestive properties. Graphical summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/542047v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@4fcd61org.highwire.dtl.DTLVardef@134c371org.highwire.dtl.DTLVardef@10b5d78org.highwire.dtl.DTLVardef@1257d19_HPS_FORMAT_FIGEXP M_FIG C_FIG

Isopentyldiol (IPDO) is an important raw material in cosmetic industry. So far IPDO is exclusively produced through chemical synthesis. Growing interest in natural personal care products has inspired the quest to develop a bio-based process. We previously reported a biosynthetic route that produces IPDO via extending leucine catabolism (route A), the efficiency of which, however, is not satisfactory. To address this issue, we computational designed a novel non-natural IPDO synthesis pathway (Route B) using RetroPath RL, the state of art tool for bio-retrosynthesis based on Artificial Intelligence methods. We compared this new pathway with route A and another two intuitively designed routes for IPDO biosynthesis from various aspects. Route B, which exhibits the highest thermodynamic driving force, least non-native reaction steps and lowest energy requirements appeared to hold the greatest potential for IPDO production. All three newly designed routes were then implemented in E. coli BL21(DE3) strain. Results show that the computationally designed route B can produce 2.2 mg/L IPDO from glucose, whereas no IPDO production from routes C and D. These results highlight the importance and usefulness of in silico design and comprehensive evaluation of the potential efficiencies of candidate pathways in constructing novel non-natural pathways for the production of biochemicals.

Our work presented here showed that MelB can be crystallized in the conditions as similar as that of other membrane transporter protein of known structure. To identify a rigid protein by modifying the protein structure is the critical factor for facilitating MelB crystallization. It is necessary to perform extensive crystallization screens to obtain crystals. MelB-MelB interaction in the DDM containing solution will be affect by protein preparation, which may lead to reduce in reproducibility of crystallization experiment. Using a detergent mixture is essential for improve protein contact in the crystals, then improve crystallizability. R149C MelB crystal can be obtained in DDM, but these crystals were only diffracted to about 8Å resolution limit. MelB wide type crystal also can be obtained from the condition as that of R149C mutant, but the resolution is weaker than that of mutant. Although MelB and other transporters of known structure share common feature of the crystallization, the emphasis was as much on the protein itself, as it was on detergent type or efficient screening and refinement of the crystallization conditions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

The global pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection confers great threat to the public health. Human breastmilk is an extremely complex with nutritional composition to nourish infants and protect them from different kinds of infection diseases and also SARS-CoV-2 infection. Previous studies have found that breastmilk exhibited potent antiviral activity against SARS-CoV-2 infection. However, it is still unknown which component(s) in the breastmilk is responsible for its antiviral activity. Here, we identified Lactoferrin (LF), MUC1 and -Lactalbumin (-LA) from human breastmilk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and in vitro confirmation that inhibited SARS-CoV-2 infection and analyzed their antiviral activity using the SARS-CoV-2 pseudovirus system and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP) in the Huh7.5, Vero E6 and Caco-2-N cell lines. Additionally, we found that LF and MUC1 could inhibit viral attachment, entry and post-entry replication, while -LA just inhibit viral attachment and entry. Importantly, LF, MUC1 and -LA possess potent antiviral activities towards not only wild-type but also variants such as B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.1 (kappa). Moreover, LF from other species (e.g., bovine and goat) is still capable of blocking viral attachment to cellular heparan sulfate. Taken together, our study provided the first line of evidence that human breastmilk components (LF, MUC1 and -LA) are promising therapeutic candidates warranting further development or treatingVID-19 given their exceedingly safety levels.

Several cardiac glycosides, including digoxin, digitoxin, and proscillaridin A, have been originally identified as cardiomyocyte modulators and are currently being investigated for their anticancer properties. These cardiac glycosides are generally classified into cardenolides and bufadienolides, which bear butenolide and pyrone D-ring functionality, respectively, and have exhibited remarkable in vitro toxicity in various cancerous cell lines. As simple modifications on steroidal small molecules have demonstrated success in augmenting bioavailability or enhancing downstream biological activities, we sought to prepare synthetic analogs of proscillaridin A, a bufadienolide isolated from the genus Scilla. We synthesized two novel analogs of proscillaridin A bearing acetate esters or a dimethyl ketal to investigate how strategies of ketalization or acetylation of the A-ring allylic glycoside might alter its anticancer properties. The antiproliferative activity of these compounds was evaluated alongside proscillaridin A and two model cardiac glycosides, digoxin and digitoxin, across several colorectal and liver cancer cell lines. Through a diverse panel of cell viability and cytotoxicity experiments, reporter assays, and cell cycle and protein marker analysis by flow cytometry, we find that ketalization of the glycan of proscillaridin A provides similar, and in some cases enhanced, in vitro potency. This study establishes the foundation for current and further in vitro and in vivo evaluation of glycan analogs of proscillaridin A.

The Notch signalling pathway is highly evolutionarily conserved and regulates differentiation and homeostasis in most organs. Given the critical role of Notch signalling for normal development, dysregulated Notch signalling is frequently linked to pathogenesis of disease and cancer. Hence, developing Notch-targeting therapeutics is warranted but has been challenging and Notch inhibitors have not yet reached broad clinical use. In this report, we identify potential Notch inhibitors, using a novel cell-based Notch reporter system for unbiased screening of compounds reducing Notch signalling. A library of 37.966 small organic compounds was screened for inhibitor candidates, followed by a counter screen to eliminate {gamma}-secretase inhibitor-like compounds and an orthogonal screen based on the role of Notch signalling in myogenic differentiation. This triage led to the identification of five Notch inhibitor candidate hits with different chemical backbones and unrelated to previous Notch antagonists. One candidate hit showed structural similarities to dihydroorotate dehydrogenase (DHODH) inhibitors, and we provide evidence that inhibition of DHODH activity reduces Notch signalling. In conclusion, our data support the notion that DHODH inhibition may be an interesting avenue to explore for the development of novel Notch inhibitors.

Targeting apoptosis is a promising approach to inhibit the abnormal cell proliferation of cancer progression. Existing anti-apoptotic drugs, many derived from chemical substances, have often failed to combat cancer development and progression. Therefore, identification of apoptosis-inducing anticancer agents from plant-derived sources has become a key aim in cancer research. The present study was designed to explore the regulation of apoptosis by Tabebuia pallida (T. pallida) using an Ehrlich Ascites Carcinoma (EAC) mouse model and compositional analysis by LC-ESI-MS/MS. Dried and powdered T. pallida leaves (TPL), stem bark (TPSB), root bark (TPRB) and flowers (TPF) were extracted with 80% methanol. Using cultured EAC cells and EAC-bearing mice with and without these extracts, anticancer activities were studied by assessing cytotoxicity and tumor cell growth inhibition, changes in life span of mice, and hematological and biochemical parameters. Apoptosis was analyzed by microscopy and expression of selected apoptosis-related genes (Bcl-2, Bcl-xL, NF{kappa}-B, PARP-1, p53, Bax, caspase-3 and -8) using RT-PCR. LC-ESI-MS analysis was performed to identify the major compounds from the most active extracts. In EAC mice compared with untreated controls, the TPL extract exhibited the highest cytotoxicity with significant tumor cell growth inhibition (p< 0.001), reduced ascites by body weight (p< 0.01), increased the life span (p<0.001), normalized blood parameters (RBC/WBC counts), and increased the levels of superoxide dismutase and catalase. TPL-treated EAC cells showed apoptotic characteristics of membrane blebbing, chromatin condensation and nuclear fragmentation, and caspase-3 activation, compared with untreated EAC cells. Moreover, annexin V-FITC and propidium iodide signals were greatly enhanced in response to TPL treatment, indicating apoptosis induction. Pro- and anti-apoptotic signaling after TPL treatment demonstrated up-regulated p53, Bax and PARP-1, and down-regulated NF{kappa}-B, Bcl-2 and Bcl-xL expression, suggesting that TPL shifts the balance of pro- and anti-apoptotic genes towards cell death. LC-ESI-MS data of TPL showed a mixture of glycosides, lapachol, and quercetin antioxidant and its derivatives that were significantly linked to cancer cell targets. In conclusion, the TPL extract of T. pallida possesses significant anticancer activity. The tumor suppressive mechanism is due to apoptosis induced by activation of antioxidant enzymes and caspases and mediated by a change in the balance of pro- and anti-apoptotic genes that promotes cell death. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/426226v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1112faaorg.highwire.dtl.DTLVardef@1505d1dorg.highwire.dtl.DTLVardef@1115a2aorg.highwire.dtl.DTLVardef@36a915_HPS_FORMAT_FIGEXP M_FIG C_FIG

Human insulin (HI) is an essential protein hormone and its biological activity mostly depends on folded and active conformation in the monomeric state. The present investigation established that Coomassie Brilliant Blue G-250 (CBBG), a small multicyclic hydroxyl compound can reversibly bind to the hormonal protein dimer and maintained most of -helical folds crucial for biological function of the enzyme. The solution-state 1D NMR and isothermal calorimetric analysis showed a sub-micromolar binding affinity of the molecule to HI. 2D NOESY NMR established that the HI dimer undergoes residue level local conformational change upon binding to CBBG. The chemical shift perturbation and the NOE parameters of active protons of amino acid residues throughout the polypeptides further suggested that CBBG upon binding the protein stabilize -helixes of both the A and B subunits of the hormonal protein. The changes in Gibbs free energy ({triangleup}G) of the binding was of ~-11.1 kcal/mol and suggested a thermodynamically favourable process. The changes in enthalpy ({triangleup}H) and entropy term (T{triangleup}S) were -57.2 kcal/mol and -46.1 kcal/mol, respectively. The negative changes in entropy and the NOE transfer effectiveness of several residues in the presence of CBBG molecules indicated that the binding was an enthalpy driven favourable equilibrium process. The NMR-based atomic resolution data and molecular docking studies confirmed that the CBBG binds to HI at the dimeric stage and prevents the availability of the crucial residue segments that partake directly in further oligomerization and subsequent fibrillation. Extended computational analysis based on chemical shift perturbation of protons of active residues further established receptor-ligand based pharmacophore model comprised of 5 hydrophobic and a hydrogen bond acceptor features that can anchor the residues at the A and B chains of HI and inhibit the partial unfolding and hydrophobic collapse to nucleate the fibrillation. Taken together, the results demonstrated that CBBG and their close analogues might be useful to develop a formulation that will maintain the active and functional form of the hormonal protein for a significantly longer time. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/267799v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b2c363org.highwire.dtl.DTLVardef@39b7b6org.highwire.dtl.DTLVardef@14c4d71org.highwire.dtl.DTLVardef@704f00_HPS_FORMAT_FIGEXP M_FIG C_FIG

Poison frogs (Dendrobatoidea) are characterized by the great diversity of alkaloids discovered in their skin. However, most of these alkaloids have been found in brightly colored species and there is a wide lack of knowledge of alkaloid profiles in the less colorful species. Previous finding of paralytic tetrodotoxins (TTXs) in only two cryptically colored species from the genus Colostethus, establishes the unique occurrence of hydrophilic alkaloids in the superfamily Dendrobatoidea. Unpublished results using extracts from Colostethus imbricolus, demonstrated that this species contains paralysis-producing substances, after intraperitoneal injection of mice. To analyze their skin metabolites and to determine if they correspond to TTX, or TTX analogues, we have employed a TTX-targeted separation in normal phase gradient, and an untargeted profiling in reversed-phase gradient. After performing both analyses, neither TTX nor TTX-analogues were detected in C. imbricolus. In contrast, other metabolites were separated, allowing the extraction of 76 adducts common to both analyses, being 33 of them tentatively annotated as amphibian alkaloids, eight as amphibian metabolites different from alkaloids and 25 that matched with natural products from the DNP. A total of 10 common molecular formulas remained non-annotated. The absence of MS/MS spectra for these adducts requires their structures to be confirmed in future analyses, following the completion of targeted MS/MS acquisition. After analyzing the inter-individual variation of six specimens, it was demonstrated that the skin metabolome differs between males and females of C. imbricolus. Our results lead us to conclude that TTX is not the only paralyzing compound in dendrobatid frogs and that more work should be undergone to identify this phenomenon. A notable additional outcome of this study is the first successful separation of TTX on an SB-CN column using a normal-phase gradient, enabling a novel method for TTX-targeted separation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/655531v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@dc4cf1org.highwire.dtl.DTLVardef@13f816eorg.highwire.dtl.DTLVardef@4a6ed6org.highwire.dtl.DTLVardef@b2b323_HPS_FORMAT_FIGEXP M_FIG C_FIG

In pursuit of isolating novel anticancer proteases from environmental microbial isolates, we have purified and identified an extracellular metallo-protease from Bacillus altitudinis named Peptidase M84. This protease selectively triggered apoptosis in human ovarian adenocarcinoma cells (PA-1, SKOV3) and mouse ovarian carcinoma cells (ID8), in addition to exhibiting no significant effect on normal human epithelial ovarian cell (IOSE) and mouse peritoneal macrophage (PEM[FE]) cell viabilities. Protease activated receptor-1 (PAR-1); a GPCR which is reported to be overexpressed in ovarian cancer cells was identified as a novel target of Peptidase M84. We observed that Peptidase M84 induced PAR-1 overexpression along with activating its downstream signalling effectors NF{kappa}B and MAPK to promote excessive reactive oxygen species (ROS) generation in ovarian cancer cells. This disrupted mitochondrial membrane potential, allowed cytosolic release of mitochondrial cytochrome c, increased the Bax (pro-apoptotic) to Bcl-2 (anti-apoptotic) ratio and promoted DNA damage to evoke apoptotic death of the ovarian cancer cells. Peptidase M84 also reduced nuclear ki-67 expression in these malignant cells to render an anti-proliferative role. In in vivo set-up, weekly intraperitoneal administration of Peptidase M84 (12 {micro}g/kg body-weight) in the ID8 mice model significantly diminished ascitic fluid accumulation through induction of oxidative stress, increasing murine survival rates by 60%. Collectively, our in vitro and in vivo findings suggested that Peptidase M84 triggered PAR-1 mediated oxidative stress to act as an apoptosis inducer in ovarian cancer cells. This established Peptidase M84 as a promising drug candidate for receptor mediated targeted-therapy of ovarian cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/556500v2_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@6b0a19org.highwire.dtl.DTLVardef@1640510org.highwire.dtl.DTLVardef@1888b49org.highwire.dtl.DTLVardef@1708114_HPS_FORMAT_FIGEXP M_FIG C_FIG

Neurodegenerative diseases (NDDs) are currently raising their prevalences and new preclinical low-cost investigations of drug design are urging. NDDs encompass a wide range of disorders, including Alzheimers, Parkinsons, ALS and others, many of which share mitochondrial dysfunction as a common pathological feature. As such, targeting mitochondrial metabolism has emerged as a promising therapeutic strategy. However, while rodent models are widely used in NDD research, they are costly and time-consuming, raising the need to consider other alternatives to accelerate the search for novel therapies. In this line, zebrafish (Danio rerio) have gained outstanding popularity as a valuable option. This systematic review aims to provide an extensive overview about the current strategies that use zebrafish assays to investigate modulations of mitochondrial function as new therapies against NDDs. The review was performed following an electronic search of different databases (PubMed, Embase, Scopus and Web of Science) after the PRISMA procedure. Articles published in the English language were identified and screened based on the keywords used: mitochondrial metabolism, therapy, neurodegenerative diseases and zebrafish. Following 176 entries, exclusion criteria reduced the record to 34 final studies. Overall, we found that these studies investigate 37 compounds: 24 natural, 6 semisynthetic, 5 synthetic and 2 compounds of not-determined origin; to ameliorate 9 prevalent diseases: ARSACS, Alzheimers, Parkinsons, Huntingtons diseases, Leigh and Wolfram syndromes, Amyotrophic lateral sclerosis, Limb - girdle muscular dystrophy 2G and hyperglycemia-associated amnesia. Additionally, a meta-analysis of these compounds and their gene interactions provides insights into their mechanisms of action and advances our understanding of NDDs, and furnishes us with a powerful tool to predictive potential new drugs or to repurpose existing ones. To conclude, this systematic review suggests that zebrafish have become an effective model for screening potential drugs for NDDs with symptomatology difficult to replicate in rodent models. Moreover, the use of computational tools is also emphasized as a promising strategy to guide therapeutic discovery more efficiently, reducing both time and costs, in developing treatments for NDDs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/710294v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@18893a1org.highwire.dtl.DTLVardef@1943a12org.highwire.dtl.DTLVardef@709146org.highwire.dtl.DTLVardef@51a488_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cancer research has made significant progress in recent years, particularly with the application of new omics technologies. By addressing proteomics, it is possible to gather vast amounts of data that offer a comprehensive view of the molecular mechanisms involved in potential treatments. This research aims to use label-free quantitative proteomics coupled with pathways and networks-based bioinformatics analysis to uncover the molecular mechanisms of the natural alkylphenol Gibbilimbol B isolated from P. eriopodon. Dynamic proteomic profiling of breast tumor cell lines treated with Gibbilimbol B showed the impact of several molecular pathways in a time and cell-dependent manner. The activation of the apoptotic pathway was confirmed for both MCF-7 and MDA-MB-231 cells; nevertheless, the proteomic profile provided new insights into molecular mechanisms triggered by the ubiquitin-dependent protein catabolic process and the identification of P53-independent DNA damage checkpoint specific for MCF-7 cells, among others. On the contrary, in MDA-MB-231, the pattern of the protein profile was correlated with a possible response to endoplasmic reticulum stress, the activation of nuclear receptors in response to pro-inflammatory processes, and an immune response related to antigen processing. Overall, these advances provide detailed insights at the cellular level for understanding potential treatment strategies.

Ethnophamacological relevanceIn the traditional folklore medicine system, the primary uses of Ruellia tuberosa L. include as a diuretic, anti-hypertensive, antipyretic, anti-diabetic, analgesic, and gastroprotective agent. Some reports also demonstrated that it has been used to treat gonorrhea-like diseases. PurposeExploring the anti-cancer potential of the methanolic extract of Ruellia tuberosa L. flower (RTME) with special emphasis on human triple-negative breast cancer (TNBC) and investigating the possible signaling networks and regulatory pathways underlying it. MethodsPreparation of RTME and identifying the possible phytochemicals through GC-MS analysis. The anti-cancer potential of RTME was executed through in-vitro cytotoxicity assay, clonogenic assay, wound healing assay, ROS generation assay, cell cycle arrest, apoptotic nuclear morphology study, cellular apoptosis study, mitochondrial membrane potential (MMP) alteration study, protein and gene expressions alteration study. Apart from this, toxicological status and in-silico molecular docking studies were also conducted. ResultsFrom this study, it was obvious that several phytochemicals within RTME have the potential to act as anti-cancerous agents. RTME was found to exhibit significant in-vitro cytotoxicity along with a reduction in colony formation and inhibition of cell migratory potential in MDA-MB-231 cells. RTME also induced intracellular ROS, promoted G0/G1 cell cycle arrest, caused mitochondrial membrane potential (MMP) alteration, and promoted cell death. From the pro- and anti-apoptotic marker study through the western blot and the qRT-PCR analysis, it was revealed that RTME promoted the intrinsic pathway of apoptosis. Furthermore, blood parameters and histological analysis revealed that RTME doesnt exhibit any toxic effect on female Balb/C mice. Finally, an in-silico molecular docking study revealed that the three identified lead phytochemicals in RTME show strong receptor-ligand interactions with the anti-apoptotic Bcl-2 and give a clue to the possible molecular mechanism of the RTME extract. ConclusionFrom the findings, it was concluded that RTME has a significant therapeutic potential against TNBC which could be an alternative option for anti-cancer drug development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/586749v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@72780corg.highwire.dtl.DTLVardef@1276029org.highwire.dtl.DTLVardef@1a2d91org.highwire.dtl.DTLVardef@a5cb44_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPreparation of methanolic extract of Ruellia tuberosa L. flower, C_LIO_LIIdentification of phytochemicals from the methanolic extract of Ruellia tuberosa L. flower, C_LIO_LIMethanolic extract of Ruellia tuberosa L. (RTME) flower exhibited significant anti-cancer potential in triple-negative breast cancer (TNBC) cells, MDA-MB-231 through induction of intracellular ROS, G0/G1 cell cycle arrest, and apoptosis, C_LIO_LIToxicological assessments of RTME on female Balb/C mice, C_LIO_LIIn-silico assessments of lead phytochemicals with the target anti-apoptotic protein, bcl-2 C_LI

CagriSema is a fixed-dose combination of cagrilintide (an amylin analogue) and semaglutide (a GLP-1 receptor agonist), and is currently an experimental obesity drug developed by Novo Nordisk. In March, 2025, CagriSema underperformed expectations in a Phase III trial, achieving 15.7% weight loss instead of the anticipated 25%, raising concerns about its efficacy and clinical value. Given its chemical composition, the weight-loss efficacy of CagriSema is inextricably linked to the activations of GLP-1R and amylin receptors (AMYRs). With GLP-1R as an example target here, this study employs a structural biophysics-guided computational approach for the design of semaglutide analogues to enhance the activation of its receptor GLP-1R. To fully harness the therapeutic potential of GLP-1R activation, an experimental structural basis (PDB entry 4ZGM) of the GLP-1-GLP-1R interaction is essential for the design of semaglutide analogues, where site-specific missense mutations are engineered into its peptide backbone to establish additional stabilizing interactions with the extracellular domain (ECD) of GLP-1R. Specifically, this study puts forward an automated systemic natural amino acid scanning of the peptide backbone of semaglutide, where PDB entry 4ZGM was used as the structural template for high-throughput structural modeling by Modeller and ligand-receptor binding affinity (Kd) calculations by Prodigy. To sum up, this article reports a total of 564 computationally designed semaglutide analogues with improved GLP-1R ECD binding affinity. Moreover, this study proposes a concept of interfacial electrostatic scaffold comprising four salt bridges at the binding interface of GLP-1R ECD and semaglutide analogues. Drawing parallels with the continued optimization in the past century history of insulin, this article argues that the interfacial electrostatic scaffold here constitutes a robust framework for continued development of next-generation GLP-1R agonists, enabling more effective therapies for patients with diabetes and/or obesity.