Mechanism of AGT-Mediated Repair of dG-dC Cross-Links in the Drug Resistance to Chloroethylnitrosoureas: Molecular Docking, MD Simulation, and ONIOM (QM/MM) Investigation.

Chloroethylnitrosoureas (CENUs) are important chemotherapies applied in the treatment of cancer. They exert anticancer activity by inducing DNA interstrand cross-links (ICLs) via the formation of two O6-alkylguanine intermediates, O6-chloroethylguanine (O6-ClEtG) and N1,O6-ethanoguanine (N1,O6-EtG). However, O6-alkylguanine-DNA alkyltransferase (AGT), a DNA-repair enzyme, can restore the O6-alkylguanine damages and thereby obstruct the formation of ICLs (dG-dC cross-link). In this study, the inhibitory mechanism of ICL formation was investigated to elucidate the drug resistance of CENUs mediated by AGT in detail. Based on the structures of the substrate-enzyme complexes obtained from docking and MD simulations, two ONIOM (QM/MM) models with different sizes of the QM region were constructed. The model with a larger QM region, which included the substrate (O6-ClEtG or N1,O6-EtG), a water molecule, and five residues (Tyr114, Cys145, His146, Lys165, and Glu172) in the active pocket of AGT, accurately described the repairing reaction and generated the results coinciding with the experimental outcomes. The repair process consists of two sequential steps: hydrogen transfer to form a thiolate anion on Cys145 and alkyl transfer from the O6 site of guanine (the rate-limiting step). The repair of N1,O6-EtG was more favorable than that of O6-ClEtG from both kinetics and thermodynamics aspects. Moreover, the comparison of the repairing process with the formation of dG-dC cross-link and the inhibition of AGT by O6-benzylguanine (O6-BG) showed that the presence of AGT could effectively interrupt the formation of ICLs leading to drug resistance, and the inhibition of AGT by O6-BG that was energetically more favorable than the repair of O6-ClEtG could not prevent the repair of N1,O6-EtG. Therefore, it is necessary to completely eliminate AGT activity before CENUs medication to enhance the chemotherapeutic effectiveness. This work provides reasonable explanations for the supposed mechanism of AGT-mediated drug resistance of CENUs and will assist in the development of novel CENU chemotherapies and their medication strategies.

The rat acute oral toxicity of trifluoromethyl compounds (TFMs): a computational toxicology study combining the 2D-QSTR, read-across and consensus modeling methods.

All areas of the modern society are affected by fluorine chemistry. In particular, fluorine plays an important role in medical, pharmaceutical and agrochemical sciences. Amongst various fluoro-organic compounds, trifluoromethyl (CF3) group is valuable in applications such as pharmaceuticals, agrochemicals and industrial chemicals. In the present study, following the strict OECD modelling principles, a quantitative structure-toxicity relationship (QSTR) modelling for the rat acute oral toxicity of trifluoromethyl compounds (TFMs) was established by genetic algorithm-multiple linear regression (GA-MLR) approach. All developed models were evaluated by various state-of-the-art validation metrics and the OECD principles. The best QSTR model included nine easily interpretable 2D molecular descriptors with clear physical and chemical significance. The mechanistic interpretation showed that the atom-type electro-topological state indices, molecular connectivity, ionization potential, lipophilicity and some autocorrelation coefficients are the main factors contributing to the acute oral toxicity of TFMs against rats. To validate that the selected 2D descriptors can effectively characterize the toxicity, we performed the chemical read-across analysis. We also compared the best QSTR model with public OPERA tool to demonstrate the reliability of the predictions. To further improve the prediction range of the QSTR model, we performed the consensus modelling. Finally, the optimum QSTR model was utilized to predict a true external set containing many untested/unknown TFMs for the first time. Overall, the developed model contributes to a more comprehensive safety assessment approach for novel CF3-containing pharmaceuticals or chemicals, reducing unnecessary chemical synthesis whilst saving the development cost of new drugs.

Prioritization of the ecotoxicological hazard of PAHs towards aquatic species spanning three trophic levels using 2D-QSTR, read-across and machine learning-driven modelling approaches.

Polycyclic aromatic hydrocarbons (PAHs) represent a common group of environmental pollutants that endanger various aquatic organisms via various pathways. To better prioritize the ecotoxicological hazard of PAHs to aquatic environment, we used 2D descriptors-based quantitative structure-toxicity relationship (QSTR) to assess the toxicity of PAHs toward six aquatic model organisms spanning three trophic levels. According to strict OECD guideline, six easily interpretable, transferable and reproducible 2D-QSTR models were constructed with high robustness and reliability. A mechanistic interpretation unveiled the key structural factors primarily responsible for controlling the aquatic ecotoxicity of PAHs. Furthermore, quantitative read-across and different machine learning approaches were employed to validate and optimize the modelling approach. Importantly, the optimum QSTR models were further applied for predicting the ecotoxicity of hundreds of untested/unknown PAHs gathered from Pesticide Properties Database (PPDB). Especially, we provided a priority list in terms of the toxicity of unknown PAHs to six aquatic species, along with the corresponding mechanistic interpretation. In summary, the models can serve as valuable tools for aquatic risk assessment and prioritization of untested or completely new PAHs chemicals, providing essential guidance for formulating regulatory policies.

Energy Blocker Lonidamine Reverses Nimustine Resistance in Human Glioblastoma Cells through Energy Blockade, Redox Homeostasis Disruption, and O6-Methylguanine-DNA Methyltransferase Downregulation: In Vitro and In Vivo Validation.

Tumor resistance seriously hinders the clinical application of chloroethylnitrosoureas (CENUs), such as O6-methylguanine-DNA methylguanine (MGMT), which can repair O6-alkyl lesions, thereby inhibiting the formation of cytotoxic DNA interstrand cross-links (ICLs). Metabolic differences between tumor and normal cells provide a biochemical basis for novel therapeutic strategies aimed at selectively inhibiting tumor energy metabolism. In this study, the energy blocker lonidamine (LND) was selected as a chemo-sensitizer of nimustine (ACNU) to explore its potential effects and underlying mechanisms in human glioblastoma in vitro and in vivo. A series of cell-level studies showed that LND significantly increased the cytotoxic effects of ACNU on glioblastoma cells. Furthermore, LND plus ACNU enhanced the energy deficiency by inhibiting glycolysis and mitochondrial function. Notably, LND almost completely downregulated MGMT expression by inducing intracellular acidification. The number of lethal DNA ICLs produced by ACNU increased after the LND pretreatment. The combination of LND and ACNU aggravated cellular oxidative stress. In resistant SF763 mouse tumor xenografts, LND plus ACNU significantly inhibited tumor growth with fewer side effects than ACNU alone. Finally, we proposed a new "HMAGOMR" chemo-sensitizing mechanism through which LND may act as a potential chemo-sensitizer to reverse ACNU resistance in glioblastoma: moderate inhibition of hexokinase (HK) activity (H); mitochondrial dysfunction (M); suppressing adenosine triphosphate (ATP)-dependent drug efflux (A); changing redox homeostasis to inhibit GSH-mediated drug inactivation (G) and increasing intracellular oxidative stress (O); downregulating MGMT expression through intracellular acidification (M); and partial inhibition of energy-dependent DNA repair (R).

From molecular descriptors to the developmental toxicity prediction of pesticides/veterinary drugs/bio-pesticides against zebrafish embryo: Dual computational toxicological approaches for prioritization.

The escalating introduction of pesticides/veterinary drugs into the environment has necessitated a rapid evaluation of their potential risks to ecosystems and human health. The developmental toxicity of pesticides/veterinary drugs was less explored, and much less the large-scale predictions for untested pesticides, veterinary drugs and bio-pesticides. Alternative methods like quantitative structure-activity relationship (QSAR) are promising because their potential to ensure the sustainable and safe use of these chemicals. We collected 133 pesticides and veterinary drugs with half-maximal active concentration (AC50) as the zebrafish embryo developmental toxicity endpoint. The QSAR model development adhered to rigorous OECD principles, ensuring that the model possessed good internal robustness (R2 > 0.6 and QLOO2 > 0.6) and external predictivity (Rtest2 > 0.7, QFn2 >0.7, and CCCtest > 0.85). To further enhance the predictive performance of the model, a quantitative read-across structure-activity relationship (q-RASAR) model was established using the combined set of RASAR and 2D descriptors. Mechanistic interpretation revealed that dipole moment, the presence of C-O fragment at 10 topological distance, molecular size, lipophilicity, and Euclidean distance (ED)-based RA function were main factors influencing toxicity. For the first time, the established QSAR and q-RASAR models were combined to prioritize the developmental toxicity of a vast array of true external compounds (pesticides/veterinary drugs/bio-pesticides) lacking experimental values. The prediction reliability of each query molecule was evaluated by leverage approach and prediction reliability indicator. Overall, the dual computational toxicology models can inform decision-making and guide the design of new pesticides/veterinary drugs with improved safety profiles.

Computer-Aided Rational Design Strategy to Improve the Thermal Stability of Alginate Lyase AlyMc.

Alginate lyase degrades alginate by the ß-elimination mechanism to produce unsaturated alginate oligosaccharides (UAOS), which have better bioactivities than saturated AOS. Enhancing the thermal stability of alginate lyases is crucial for their industrial applications. In this study, a feasible and efficient rational design strategy was proposed by combining the computer-aided ΔΔG value calculation with the B-factor analysis. Two thermal stability-enhanced mutants, Q246V and K249V, were obtained by site-directed mutagenesis. Particularly, the t1/2, 50 °C for mutants Q246V and K249V was increased from 2.36 to 3.85 and 3.65 h, respectively. Remarkably, the specific activities of Q246V and K249V were enhanced to 2.41- and 2.96-fold that of alginate lyase AlyMc, respectively. Structural analysis and molecular dynamics simulations suggested that mutations enhanced the hydrogen bond networks and the overall rigidity of the molecular structure. Notably, mutant Q246V exhibited excellent thermal stability among the PL-7 alginate lyase family, especially considering the heightened enzymatic activity. Moreover, the rational design strategy used in this study can effectively improve the thermal stability of enzymes and has important significance in advancing applications of alginate lyase.

Recent advances in chemometric modelling of inhibitors against SARS-CoV-2.

The outbreak of the novel coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused great harm to all countries worldwide. This disease can be prevented by vaccination and managed using various treatment methods, including injections, oral medications, or aerosol therapies. However, the selection of suitable compounds for the research and development of anti-SARS-CoV-2 drugs is a daunting task because of the vast databases of available compounds. The traditional process of drug research and development is time-consuming, labour-intensive, and costly. The application of chemometrics can significantly expedite drug R&D. This is particularly necessary and important for drug development against pandemic public emergency diseases, such as COVID-19. Through various chemometric techniques, such as quantitative structure-activity relationship (QSAR) modelling, molecular docking, and molecular dynamics (MD) simulations, compounds with inhibitory activity against SARS-CoV-2 can be quickly screened, allowing researchers to focus on the few prioritised candidates. In addition, the ADMET properties of the screened candidate compounds should be further explored to promote the successful discovery of anti-SARS-CoV-2 drugs. In this case, considerable time and economic costs can be saved while minimising the need for extensive animal experiments, in line with the 3R principles. This paper focuses on recent advances in chemometric modelling studies of COVID-19-related inhibitors, highlights current limitations, and outlines potential future directions for development.

A Tween-80 modified hypoxia/esterase dual stimulus-activated nanomicelle as a delivery platform for carmustine - Design, synthesis, and biological evaluation.

As a clinical anti-glioma agent, the therapeutic effect of carmustine (BCNU) was largely decreased because of the drug resistance mediated by O6-alkylguanine-DNA alkyltransferase (AGT) and the blood-brain barrier (BBB). To overcome these obstacles, we synthesized a BCNU-loaded hypoxia/esterase dual stimulus-activated nanomicelle, abbreviated as T80-HACB/BCNU NPs. In this nano-system, Tween 80 acts as the functional coating on the surface of the micelle to facilitate transport across the BBB. Hyaluronic acid (HA) with active tumor-targeting capability was linked with the hypoxia-sensitive AGT inhibitors (O6-azobenzyloxycarbonyl group) via an esterase-activated ester bond. The obtained T80-HACB/BCNU NPs had an average particle size of 232.10 ± 10.66 nm, the zeta potential of -18.13 ± 0.91 mV, and it showed high drug loading capacity, eximious biocompatibility and dual activation of hypoxia/esterase drug release behavior. The obtained T80-HACB/BCNU NPs showed enhanced cytotoxicity against hypoxic T98G and SF763 cells with IC50 at 132.2 µM and 133.1 µM, respectively. T80 modification improved the transportation of the micelle across an in vitro BBB model. The transport rate of the T80-HACB/Cou6 NPs group was 12.37 %, which was 7.6-fold (p<0.001) higher than the micelle without T80 modification. T80-HACB/BCNU NPs will contribute to the development of novel CENUs chemotherapies with high efficacy.

Quality changes of whitespotted conger (Conger myriaster) based physicochemical changes and label-free proteomics analysis during frozen storage.

Whitespotted conger (Conger myriaster) muscle proteins were susceptible to oxidative denaturation during frozen storage. The objective of this study was to investigate the alterations in quality through physicochemical analysis and proteomics after whitespotted conger stored at temperatures of -18 °C and -60 °C. The microstructural observation revealed the noticeable variations such as increased interstitial space and fractured muscle fibre with extension of frozen storage time, and the muscle fibre of whitespotted conger stored at -60 °C were more intact than those stored at -18 °C. The raised TVB-N value indicated that the freshness of whitespotted conger decreased during 120-day frozen storage period. Analysis of myofibrillar protein content and SDS-PAGE demonstrated that compared to -18 °C, lower storage temperature (-60 °C) could better maintain the structure of whitespotted conger muscle by inhibiting protein degradation and oxidation. To reveal the mechanism of protein degradation, label-free quantitative proteomic analysis was performed through LC-MS/MS. The structural proteins including domain-associated proteins and actin-related proteins were up-regulated during frozen storage, but the phosphoglycerate kinase, phosphoglycerate mutase, and fructose-bisphosphate aldolase were down-regulated. Storage at -18 °C accelerated the up- or down-regulation of those differentially abundant proteins. According to KEGG analysis, up- or down-regulated pathways such as glycolysis/gluconeogenesis, carbon metabolism, biosynthesis of amino acids, and calcium signalling pathway mainly accounted for the protein degradation and quality reduction of whitespotted conger at low temperature. These results provided a theoretical basis for improving the quality stability of whitespotted conger during frozen storage.