Synthesis, structural characterization, analytical profiling, and application to authentic samples of synthesized Phase I metabolites of the synthetic cannabinoid 5F-MDMB-PICA.

5F-MDMB-PICA, an indole-type synthetic cannabinoid (SC), was classified illicit globally in 2020. Although the extensive metabolism of 5F-MDMB-PICA in the human body warrants the development of robust analytical methods for metabolite detection and quantification, a current lack of reference standards for characteristic metabolites hinders such method creation. This work described the synthesis of 18 reference standards for 5F-MDMB-PICA and its possible Phase I metabolites, including three hydroxylated positional isomers R14 to R16. All the compounds were systematic characterized via nuclear magnetic resonance, Fourier transform infrared spectroscopy, and high-resolution mass spectrometry. Furthermore, two methods were developed for the simultaneous detection of all standards using liquid chromatography-tandem mass spectrometry and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. By comparison with authentic samples, R17 was identified as a suitable urine biomarker for 5F-MDMB-PICA uptake.

Enantioselective Toxicity and Potential Endocrine-Disruptive Effects of the Insecticides Flufiprole and Ethiprole on Danio rerio.

Phenylpyrazole insecticides are widely used as chiral pesticides. However, the enantioselective toxicity and potential endocrine-disrupting effects of these insecticides on aquatic organisms remain unclear. Herein, the enantioselective toxicity and potential endocrine-disrupting effects of flufiprole and ethiprole were investigated by using zebrafish embryos/larvae as a model. The acute toxicity of R-flufiprole and R-ethiprole toward zebrafish embryos and larvae was 1.8-3.1-fold higher than that of the S-configuration. Additionally, R-flufiprole and R-ethiprole had a greater effect on the expression of genes related to the hypothalamus-pituitary-gonad axis in zebrafish compared with the S-configuration. Nevertheless, both S-flufiprole and S-ethiprole exhibited a greater interference effect on the expression of genes related to the hypothalamus-pituitary-thyroid axis and a greater teratogenic effect on zebrafish than the R-configuration. Thus, this study demonstrates that both flufiprole and ethiprole exhibit enantioselective acute toxicity and developmental toxicity toward zebrafish. Furthermore, those pesticides potentially possess enantioselective endocrine-disrupting effects.

Syntheses of Cannabinoid Metabolites: Ajulemic Acid and HU-210.

Cannabinoid metabolites have been reported to be more potent than their parent compounds. Among them, ajulemic acid (AJA) is a side-chain analog of Δ9-THC-11-oic acid, which would be a good template structure for the discovery of more potent analogues. Herein, we optimized the key allylic oxidation step to introduce the C-11 hydroxy group with a high yield. A series of compounds was prepared with this condition applied including HU-210, 11-nor-Δ8-tetrahydrocannabinol (THC)-carboxylic acid and Δ9-THC-carboxylic acid.

The Synthesis of Biphasic Metabolites of Carfentanil.

Carfentanil is an ultra-potent synthetic opioid. The Russian police force used both carfentanil and remifentanil to resolve a hostage incident in Moscow. This reported use sparked an interest in the pharmacology and toxicology of carfentanil in the human body, and data on its metabolites were later published. However, there have been few studies on the synthesis of carfentanil metabolites, and biological extraction has also put forward large uncertainty in subsequent studies. The aim of the present study is to investigate the synthesis of biphasic metabolites that are unique to carfentanil. The purpose was to produce corresponding metabolites conveniently, quickly, and at low cost that can be used for comparison with published structures and to confirm the administration of carfentanil.

Iterative Knowledge-Based Scoring Function for Protein-Ligand Interactions by Considering Binding Affinity Information.

Scoring functions for protein-ligand interactions play a critical role in structure-based drug design. Owing to the good balance between general applicability and computational efficiency, knowledge-based scoring functions have obtained significant advancements and achieved many successes. Nevertheless, knowledge-based scoring functions face a challenge in utilizing the experimental affinity data and thus may not perform well in binding affinity prediction. Addressing the challenge, we have proposed an improved version of the iterative knowledge-based scoring function ITScore by considering binding affinity information, which is referred to as ITScoreAff, based on a large training set of 6216 protein-ligand complexes with both structures and affinity data. ITScoreAff was extensively evaluated and compared with ITScore, 33 traditional, and 6 machine learning scoring functions in terms of docking power, ranking power, and screening power on the independent CASF-2016 benchmark. It was shown that ITScoreAff obtained an overall better performance than the other 40 scoring functions and gave an average success rate of 85.3% in docking power, a correlation coefficient of 0.723 in scoring power, and an average rank correlation coefficient of 0.668 in ranking power. In addition, ITScoreAff also achieved the overall best screening power when the top 10% of the ranked database were considered. These results demonstrated the robustness of ITScoreAff and its improvement over existing scoring functions.

Heptanoic and medium branched-chain fatty acids as anaplerotic treatment for medium chain acyl-CoA dehydrogenase deficiency.

Triheptanoin (triheptanoylglycerol) has shown value as anaplerotic therapy for patients with long chain fatty acid oxidation disorders but is contraindicated in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In search for anaplerotic therapy for patients with MCAD deficiency, fibroblasts from three patients homozygous for the most common mutation, ACADMG985A/G985A, were treated with fatty acids hypothesized not to require MCAD for their metabolism, including heptanoic (C7; the active component of triheptanoin), 2,6-dimethylheptanoic (dMC7), 6-amino-2,4-dimethylheptanoic (AdMC7), or 4,8-dimethylnonanoic (dMC9) acids. Their effectiveness as anaplerotic fatty acids was assessed in live cells by monitoring changes in cellular oxygen consumption rate (OCR) and mitochondrial protein lysine succinylation, which reflects cellular succinyl-CoA levels, using immunofluorescence (IF) staining. Krebs cycle intermediates were also quantitated in these cells using targeted metabolomics. The four fatty acids induced positive changes in OCR parameters, consistent with their oxidative catalysis and utilization. Increases in cellular IF staining of succinylated lysines were observed, indicating that the fatty acids were effective sources of succinyl-CoA in the absence of media glucose, pyruvate, and lipids. The ability of MCAD deficient cells to metabolize C7 was confirmed by the ability of extracts to enzymatically utilize C7-CoA as substrate but not C8-CoA. To evaluate C7 therapeutic potential in vivo, Acadm-/- mice were treated with triheptanoin for seven days. Dose dependent increase in plasma levels of heptanoyl-, valeryl-, and propionylcarnitine indicated efficient metabolism of the medication. The pattern of the acylcarnitine profile paralleled resolution of liver pathology including reversing hepatic steatosis, increasing hepatic glycogen content, and increasing hepatocyte protein succinylation, all indicating improved energy homeostasis in the treated mice. These results provide the impetus to evaluate triheptanoin and the medium branched chain fatty acids as potential therapeutic agents for patients with MCAD deficiency.

Messenger RNA rescues medium-chain acyl-CoA dehydrogenase deficiency in fibroblasts from patients and a murine model.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid ß-oxidation (FAO) in humans. Patients exhibit clinical episodes often associated with fasting. Symptoms include hypoketotic hypoglycemia and Reye-like episodes. With limited treatment options, we explored the use of human MCAD (hMCAD) mRNA in fibroblasts from patients with MCAD deficiency to provide functional MCAD protein and reverse the metabolic block. Transfection of hMCAD mRNA into MCAD- deficient patient cells resulted in an increased MCAD protein that localized to mitochondria, concomitant with increased enzyme activity in cell extracts. The therapeutic hMCAD mRNA-lipid nanoparticle (LNP) formulation was also tested in vivo in Acadm-/- mice. Administration of multiple intravenous doses of the hMCAD mRNA-LNP complex (LNP-MCAD) into Acadm-/- mice produced a significant level of MCAD protein with increased enzyme activity in liver, heart and skeletal muscle homogenates. Treated Acadm-/- mice were more resistant to cold stress and had decreased plasma levels of medium-chain acylcarnitines compared to untreated animals. Furthermore, hepatic steatosis in the liver from treated Acadm-/- mice was reduced compared to untreated ones. Results from this study support the potential therapeutic value of hMCAD mRNA-LNP complex treatment for MCAD deficiency.

Synthetic mRNA rescues very long-chain acyl-CoA dehydrogenase deficiency in patient fibroblasts and a murine model.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an inborn error of long chain fatty acid ß-oxidation (FAO) with limited treatment options. Patients present with heterogeneous clinical phenotypes affecting predominantly heart, liver, and skeletal muscle. While VLCAD deficiency is a systemic disease, restoration of liver FAO has the potential to improve symptoms more broadly due to increased total body ATP production and reduced accumulation of potentially toxic metabolites. We explored the use of synthetic human VLCAD (hVLCAD) mRNA and lipid nanoparticle encapsulated hVLCAD mRNA (LNP-VLCAD) to generate functional VLCAD enzyme in patient fibroblasts derived from VLCAD deficient patients, mouse embryonic fibroblasts, hepatocytes isolated from VLCAD knockout (Acadvl-/-) mice, and Acadvl-/- mice to reverse the metabolic effects of the deficiency. Transfection of all cell types with hVLCAD mRNA resulted in high level expression of protein that localized to mitochondria with increased enzyme activity. Intravenous administration of LNP-VLCAD to Acadvl-/- mice produced a significant amount of VLCAD protein in liver, which declined over a week. Treated Acadvl-/- mice showed reduced hepatic steatosis, were more resistant to cold stress, and accumulated less toxic metabolites in blood than untreated animals. Results from this study support the potential for hVLCAD mRNA for treatment of VLCAD deficiency.

Holo Protein Conformation Generation from Apo Structures by Ligand Binding Site Refinement.

An important part in structure-based drug design is the selection of an appropriate protein structure. It has been revealed that a holo protein structure that contains a well-defined binding site is a much better choice than an apo structure in structure-based drug discovery. Therefore, it is valuable to obtain a holo-like protein conformation from apo structures in the case where no holo structure is available. Meeting the need, we present a robust approach to generate reliable holo-like structures from apo structures by ligand binding site refinement with restraints derived from holo templates with low homology. Our method was tested on a test set of 32 proteins from the DUD-E data set and compared with other approaches. It was shown that our method successfully refined the apo structures toward the corresponding holo conformations for 23 of 32 proteins, reducing the average all-heavy-atom RMSD of binding site residues by 0.48 Å. In addition, when evaluated against all the holo structures in the protein data bank, our method can improve the binding site RMSD for 14 of 19 cases that experience significant conformational changes. Furthermore, our refined structures also demonstrate their advantages over the apo structures in ligand binding mode predictions by both rigid docking and flexible docking and in virtual screening on the database of active and decoy ligands from the DUD-E. These results indicate that our method is effective in recovering holo-like conformations and will be valuable in structure-based drug discovery.

One-click device for rapid visualization and extraction of latent evidence through multi-moding light source integration and light-guiding technology.

Visualizing latent evidence at a crime scene has gained popularity in the field of forensic science during the past few years. Thus, this study designs and develops a one-click device for the rapid visualization and extraction of latent evidence through multimodal light source integration and light-guiding technology. Our device exhibits multispectral and angle timing functions for storing the captured evidence images. Furthermore, the geometric registration, feature extraction, feature optimization, and feature integration of the evidence images are processed by a backend system, and the images are then presented. Overall, this study enhances the standard and the technical content of evidence extraction and simplifies the evidence extraction process. In addition to the rapid handling of the scenes captured at a crime scene, the one-click device has other notable advantages, such as fast imaging, portability, being independent of the environmental conditions and the operator's technical capabilities, and zero pollution to ensure the repeatability of material evidence extraction. Compared with the original optical forensics equipment, the spectrum and angle of our system are more extensive.

Synthesis and biological evaluation of novel all-hydrocarbon cross-linked aza-stapled peptides.

Novel all-hydrocarbon cross-linked aza-stapled peptides were designed and synthesized for the first time by ring-closing metathesis between two aza-alkenylglycine residues. Three aza-stapled peptidic analogues based on the peptide dual inhibitor of p53-MDM2/MDMX interactions were synthesized and screened for biological activities. Among the three aza-stapled peptides, aSPDI-411 displayed increased anti-tumor activity, binding affinities to both MDM2 and MDMX, and cell membrane permeability compared to its linear peptide counterpart.

miR-182-5p attenuates Schistosoma japonicum-induced hepatic fibrosis by targeting tristetraprolin.

Egg granuloma formation in the liver is the main pathological lesion caused by Schistosoma japonicum infection, which generally results in liver fibrosis and may lead to death in advanced patients. MicroRNAs (miRNAs) regulate the process of liver fibrosis, but the putative function of miRNAs in liver fibrosis induced by S. japonicum infection is largely unclear. Here, we detect a new miRNA, miR-182-5p, which shows significantly decreased expression in mouse livers after stimulation by soluble egg antigen (SEA) of S. japonicum or S. japonicum infection. Knockdown or overexpression of miR-182-5p in vitro causes the increased or decreased expression of tristetraprolin (TTP), an important immunosuppressive protein in the process of liver fibrosis. Furthermore, knockdown of miR-182-5p in vivo upregulates TTP expression and significantly alleviates S. japonicum-induced hepatic fibrosis. Our data demonstrate that downregulation of miR-182-5p increases the expression of TTP in mouse livers following schistosome infection, which leads to destabilization of inflammatory factor mRNAs and attenuates liver fibrosis. Our results uncover fine-tuning of liver inflammatory reactions related to liver fibrosis caused by S. japonicum infection and provide new insights into the regulation of schistosomiasis-induced hepatic fibrosis.

Lysine-Tethered Stable Bicyclic Cationic Antimicrobial Peptide Combats Bacterial Infection in Vivo.

Antimicrobial peptides (AMPs) have attracted great attention as next generation antibiotics for the treatment of multidrug-resistant (MDR) bacterial infections. Poor proteolytic stability has however undermined clinical applications of AMPs. A novel peptide cyclization approach is described to enhance the in vivo antibacterial activity of AMPs. Bicyclic antimicrobial peptides were synthesized by cross-linking the ε-amino groups of three lysine residues with a 1,3,5-trimethylene benzene spacer. In a proof of principal study, four bicyclic peptides were synthesized from the cationic AMP OH-CM6. One bicyclic peptide retained strong antimicrobial activity and low toxicity but exhibited a prolonged half-life in serum. Antibacterial activity was consequently improved in vivo without renal or hepato-toxicity. The novel peptide cyclization approach represents an important tool for enhancing AMP proteolytic stability for improved treatment of bacterial infection.

Docking cyclic peptides formed by a disulfide bond through a hierarchical strategy.

MOTIVATION: Cyclization is a common strategy to enhance the therapeutic potential of peptides. Many cyclic peptide drugs have been approved for clinical use, in which the disulfide-driven cyclic peptide is one of the most prevalent categories. Molecular docking is a powerful computational method to predict the binding modes of molecules. For protein-cyclic peptide docking, a big challenge is considering the flexibility of peptides with conformers constrained by cyclization. RESULTS: Integrating our efficient peptide 3D conformation sampling algorithm MODPEP2.0 and knowledge-based scoring function ITScorePP, we have proposed an extended version of our hierarchical peptide docking algorithm, named HPEPDOCK2.0, to predict the binding modes of the peptide cyclized through a disulfide against a protein. Our HPEPDOCK2.0 approach was extensively evaluated on diverse test sets and compared with the state-of-the-art cyclic peptide docking program AutoDock CrankPep (ADCP). On a benchmark dataset of 18 cyclic peptide-protein complexes, HPEPDOCK2.0 obtained a native contact fraction of above 0.5 for 61% of the cases when the top prediction was considered, compared with 39% for ADCP. On a larger test set of 25 cyclic peptide-protein complexes, HPEPDOCK2.0 yielded a success rate of 44% for the top prediction, compared with 20% for ADCP. In addition, HPEPDOCK2.0 was also validated on two other test sets of 10 and 11 complexes with apo and predicted receptor structures, respectively. HPEPDOCK2.0 is computationally efficient and the average running time for docking a cyclic peptide is about 34 min on a single CPU core, compared with 496 min for ADCP. HPEPDOCK2.0 will facilitate the study of the interaction between cyclic peptides and proteins and the development of therapeutic cyclic peptide drugs. AVAILABILITY AND IMPLEMENTATION: http://huanglab.phys.hust.edu.cn/hpepdock/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Adaptive Contrastive Learning with Label Consistency for Source Data Free Unsupervised Domain Adaptation.

Unsupervised domain adaptation, which aims to alleviate the domain shift between source domain and target domain, has attracted extensive research interest; however, this is unlikely in practical application scenarios, which may be due to privacy issues and intellectual rights. In this paper, we discuss a more challenging and practical source-free unsupervised domain adaptation, which needs to adapt the source domain model to the target domain without the aid of source domain data. We propose label consistent contrastive learning (LCCL), an adaptive contrastive learning framework for source-free unsupervised domain adaptation, which encourages target domain samples to learn class-level discriminative features. Considering that the data in the source domain are unavailable, we introduce the memory bank to store the samples with the same pseudo label output and the samples obtained by clustering, and the trusted historical samples are involved in contrastive learning. In addition, we demonstrate that LCCL is a general framework that can be applied to unsupervised domain adaptation. Extensive experiments on digit recognition and image classification benchmark datasets demonstrate the effectiveness of the proposed method.

Visible light-promoted intermolecular cyclization/aromatization of chalcones and 2-mercaptobenzimidazoles via an EDA complex and a mechanism study.

Visible-light-promoted cyclization and aromatization of chalcones with 2-mercaptobenzimidazoles have been successfully developed to obtain diverse imidazo[2,1-b]thiazoles, and C-S and C-N bonds were constructed in one step. The reaction uses oxygen in the air as an oxidant, and the method does not need an external photocatalyst or a transition metal catalyst. The strategy features mild conditions, a simple system, readily accessible feedstocks, and a friendly environment. UV absorption spectroscopy and control experiments have shown that the reaction mechanism involves the formation of an electron-donor-acceptor (EDA) complex from thiolate anions and chalcones. In order to verify the mechanism, we studied the structure and HOMO/LUMO of the EDA complex by density functional theory (DFT) calculations. The results show that the π-π stacking between chalcones and 2-mercaptobenzimidazoles will cause a red shift of the UV absorption wavelength in the presence of Cs2CO3, and also provide a theoretical basis for the electron transfer of EDA complexes.

Efficient 3D conformer generation of cyclic peptides formed by a disulfide bond.

Cyclic peptides formed by disulfide bonds have been one large group of common drug candidates in drug development. Structural information of a peptide is essential to understand its interaction with its target. However, due to the high flexibility of peptides, it is difficult to sample the near-native conformations of a peptide. Here, we have developed an extended version of our MODPEP approach, named MODPEP2.0, to fast generate the conformations of cyclic peptides formed by a disulfide bond. MODPEP2.0 builds the three-dimensional (3D) structures of a cyclic peptide from scratch by assembling amino acids one by one onto the cyclic fragment based on the constructed rotamer and cyclic backbone libraries. Being tested on a data set of 193 diverse cyclic peptides, MODPEP2.0 obtained a considerable advantage in both accuracy and computational efficiency, compared with other sampling algorithms including PEP-FOLD, ETKDG, and modified ETKDG (mETKDG). MODPEP2.0 achieved a high sampling accuracy with an average C[Formula: see text] RMSD of 2.20 Å and 1.66 Å when 10 and 100 conformations were considered, respectively, compared with 3.41 Å and 2.62 Å for PEP-FOLD, 3.44 Å and 3.16 Å for ETKDG, 3.09 Å and 2.72 Å for mETKDG. MODPEP2.0 also reproduced experimental peptide structures for 81.35% of the test cases when an ensemble of 100 conformations were considered, compared with 54.95%, 37.50% and 50.00% for PEP-FOLD, ETKDG, and mETKDG. MODPEP2.0 is computationally efficient and can generate 100 peptide conformations in one second. MODPEP2.0 will be useful in sampling cyclic peptide structures and modeling related protein-peptide interactions, facilitating the development of cyclic peptide drugs.

Inclusion of Desolvation Energy into Protein-Protein Docking through Atomic Contact Potentials.

Protein-protein interactions are crucial in many biological processes. Therefore, determining the structure of a protein-protein complex is valuable for understanding its molecular mechanisms and developing drugs. Molecular docking is a powerful computational tool in the prediction of protein-protein complex structures, in which a scoring function with good performance is very important. In this study, we have proposed a hybrid scoring function of atomic contact-based desolvation energies and distance-dependent interatomic potentials for protein-protein interactions, named HITScorePP, where the atomic contact desolvation energies were derived using an iterative method and the distance-dependent potentials were directly taken from our ITScorePP scoring function. Integrating the hybrid scoring function into our fast Fourier transform (FFT) based HDOCK docking scheme, the updated docking program, named HDOCK2.0, significantly improved the docking performance on the 55 newly added complexes in the protein docking benchmark 5.0 and a data set of 19 antibacterial protein complexes. HDOCK2.0 was also compared with four other state-of-the-art docking programs including Rosetta, ZDOCK3.0.2, FRODOCK3.0, ATTRACT, and PatchDock and obtained the overall best performance in binding mode predictions. These results demonstrated the accuracy of our hybrid scoring function and the necessity of included desolvation effects in protein-protein docking.

TTP protects against acute liver failure by regulating CCL2 and CCL5 through m6A RNA methylation.

Tristetraprolin (TTP), an important immunosuppressive protein regulating mRNA decay through recognition of the AU-rich elements (AREs) within the 3'-UTRs of mRNAs, participates in the pathogenesis of liver diseases. However, whether TTP regulates mRNA stability through other mechanisms remains poorly understood. Here, we report that TTP was upregulated in acute liver failure (ALF), resulting in decreased mRNA stabilities of CCL2 and CCL5 through promotion of N6-methyladenosine (m6A) mRNA methylation. Overexpression of TTP could markedly ameliorate hepatic injury in vivo. TTP regulated the mRNA stabilization of CCL2 and CCL5. Interestingly, increased m6A methylation in CCL2 and CCL5 mRNAs promoted TTP-mediated RNA destabilization. Moreover, induction of TTP upregulated expression levels of WT1 associated protein, methyltransferase like 14, and YT521-B homology N6-methyladenosine RNA binding protein 2, which encode enzymes regulating m6A methylation, resulting in a global increase of m6A methylation and amelioration of liver injury due to enhanced degradation of CCL2 and CCL5. These findings suggest a potentially novel mechanism by which TTP modulates mRNA stabilities of CCL2 and CCL5 through m6A RNA methylation, which is involved in the pathogenesis of ALF.

A Sensitive and Selective Fluorescent Probe for Real-Time Detection and Imaging of Hypochlorous Acid in Living Cells.

Hypochlorous acid (HClO), a reactive oxygen species, plays an essential role in the processes of physiology and pathology via reacting with most biological molecules. The abnormal level of HClO may cause inflammation, especially arthritis. To further understand its key role in inflammation, in situ detection of HClO is necessary. Herein, a water-soluble small molecule fluorescent probe (HDI-HClO) is employed to monitor and identify trace amounts of HClO in the biological system. In the presence of HClO, the probe releases a hydroxyl group emitting strong fluorescence because of the restoration of the intramolecular charge transfer process. Furthermore, this probe displays a 150-fold fluorescence enhancement accompanied by a large Stokes shift and a lower detection limit (8.3 nM). Moreover, the probe can make a rapid response to HClO within 8 s, which provides the possibility of real-time monitoring of intracellular HClO. Based on the advantages of rapid dynamics, good water solubility, and excellent biocompatibility, this probe could effectively monitor the fluctuations of exogenous and endogenous HClO in living cells. The fluorescence imaging of HDI-HClO indicated that it is an excellent potential approach for comprehending the relationship between inflammation and HClO.