Target Identification Strategies Research Articles

RNA lipid nanoparticles as efficient in vivo CRISPR-Cas9 gene editing tool for therapeutic target validation in glioblastoma cancer stem cells

In vitro and ex-vivo target identification strategies often fail to predict in vivo efficacy, particularly for glioblastoma (GBM), a highly heterogenous tumor rich in resistant cancer stem cells (GSCs). An in vivo screening tool can improve prediction of therapeutic efficacy by considering the complex tumor microenvironment and the dynamic plasticity of GSCs driving therapy resistance and recurrence. This study proposes lipid nanoparticles (LNPs) as an efficient in vivo CRISPR-Cas9 gene editing tool for target validation in mesenchymal GSCs. LNPs co-delivering mRNA (mCas9) and single-guide RNA (sgRNA) were successfully formulated and optimized facilitating both in vitro and in vivo gene editing. In vitro, LNPs achieved up to 67 % reduction in green fluorescent protein (GFP) expression, used as a model target, outperforming a commercial transfection reagent. Intratumoral administration of LNPs in GSCs resulted in ∼80 % GFP gene knock-out and a 2-fold reduction in GFP signal by day 14. This study showcases the applicability of CRISPR-Cas9 LNPs as a potential in vivo screening tool in GSCs, currently lacking effective treatment. By replacing GFP with a pool of potential targets, the proposed platform presents an exciting prospect for therapeutic target validation in orthotopic GSCs, bridging the gap between preclinical and clinical research.

Hunting for the Intermolecular Diels-Alderase.

ConspectusThe Diels-Alder reaction is well known as a concerted [4 + 2] cycloaddition governed by the Woodward-Hoffmann rules. Since Prof. Otto Diels and his student Kurt Alder initially reported the intermolecular [4 + 2] cycloaddition between cyclopentadiene and quinone in 1928, it has been recognized as one of the most powerful chemical transformations to build C-C bonds and construct cyclic structures. This named reaction has been widely used in synthesizing natural products and drug molecules. Driven by the synthetic importance of the Diels-Alder reaction, identifying the enzyme that stereoselectively catalyzes the Diels-Alder reaction has become an intriguing research area in natural product biosynthesis and biocatalysis. With significant progress in sequencing and bioinformatics, dozens of Diels-Alderases have been characterized in microbial natural product biosynthesis. However, few are evolutionally dedicated to catalyzing an intermolecular Diels-Alder reaction with a concerted mechanism.This Account summarizes our endeavors to hunt for the naturally occurring intermolecular Diels-Alderase from plants. Our research journey started from the biomimetic syntheses of D-A-type terpenoids and flavonoids, showing that plants use both nonenzymatic and enzymatic intermolecular [4 + 2] cycloadditions to create complex molecules. Inspired by the biomimetic syntheses, we identify an intermolecular Diels-Alderase hidden in the biosynthetic pathway of mulberry Diels-Alder-type cycloadducts using a biosynthetic intermediate probe-based target identification strategy. This enzyme, MaDA, is an endo-selective Diels-Alderase and is then functionally characterized as a standalone intermolecular Diels-Alderase with a concerted but asynchronous mechanism. We also discover the exo-selective intermolecular Diels-Alderases in Morus plants. Both the endo- and exo-selective Diels-Alderases feature a broad substrate scope, but their mechanisms for controlling the endo/exo pathway are different. These unique intermolecular Diels-Alderases phylogenetically form a subgroup of FAD-dependent enzymes that can be found only in moraceous plants, explaining why this type of [4 + 2] cycloadduct is unique to moraceous plants. Further studies of the evolutionary mechanism reveal that an FAD-dependent oxidocyclase could acquire the Diels-Alderase activity via four critical amino acid mutations and then gradually lose its original oxidative activity to become a standalone Diels-Alderase during the natural evolution. Based on these insights, we designed new Diels-Alderases and achieved the diversity-oriented chemoenzymatic synthesis of D-A products using either naturally occurring or engineered Diels-Alderases.Overall, this Account describes our decade-long efforts to discover the intermolecular Diels-Alderases in Morus plants, particularly highlighting the importance of biomimetic synthesis and chemical proteomics in discovering new intermolecular Diels-Alderases from plants. Meanwhile, this Account also covers the evolutionary and catalytic mechanism study of intermolecular Diels-Alderases that may provide new insights into how to discover and design new Diels-Alderases as powerful biocatalysts for organic synthesis.

Degradation-Based Protein Profiling: A Case Study of Celastrol.

Natural products, while valuable for drug discovery, encounter limitations like uncertainty in targets and toxicity. As an important active ingredient in traditional Chinese medicine, celastrol exhibits a wide range of biological activities, yet its mechanism remains unclear. In this study, they introduced an innovative "Degradation-based protein profiling (DBPP)" strategy, which combined PROteolysis TArgeting Chimeras (PROTAC) technology with quantitative proteomics and Immunoprecipitation-Mass Spectrometry (IP-MS) techniques, to identify multiple targets of natural products using a toolbox of degraders. Taking celastrol as an example, they successfully identified its known targets, including inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PI3Kα), and cellular inhibitor of PP2A (CIP2A), as well as potential new targets such as checkpoint kinase 1 (CHK1), O-GlcNAcase (OGA), and DNA excision repair protein ERCC-6-like (ERCC6L). Furthermore, the first glycosidase degrader is developed in this work. Finally, by employing a mixed PROTAC toolbox in quantitative proteomics, they also achieved multi-target identification of celastrol, significantly reducing costs while improving efficiency. Taken together, they believe that the DBPP strategy can complement existing target identification strategies, thereby facilitating the rapid advancement of the pharmaceutical field.

Lanthanide metal-organic framework-based surface molecularly imprinted polymers ratiometric fluorescence probe for visual detection of perfluorooctanoic acid with a smartphone-assisted portable device

Perfluorooctanoic acid (PFOA) poses a threat to the environment and human health due to its persistence, bioaccumulation, and reproductive toxicity. Herein, a lanthanide metal-organic framework (Ln-MOF)-based surface molecularly imprinted polymers (SMIPs) ratiometric fluorescence probe (Eu/Tb-MOF@MIPs) and a smartphone-assisted portable device were developed for the detection of PFOA with high selectivity in real water samples. The integration of Eu/Tb MOFs as carriers not only had highly stable multiple emission signals but also prevented deformation of the imprinting cavity of MIPs. Meanwhile, the MIPs layer preserved the fluorescence of Ln-MOF and provided selective cavities for improved specificity. Molecular dynamics (MD) was employed to simulate the polymerization process of MIPs, revealing that the formation of multiple recognition sites was attributed to the establishment of hydrogen bonds between functional monomers and templates. The probe showed a good linear relationship with PFOA concentration in the range of 0.02–2.8 μM, by giving the limit of detection (LOD) of 0.98 nM. Additionally, The red-green-blue (RGB) values analysis based on the smartphone-assisted portable device demonstrated a linear relationship of 0.1–2.8 μM PFOA with the LOD of 3.26 nM. The developed probe and portable device sensing platform exhibit substantial potential for on-site detecting PFOA in practical applications and provide a reliable strategy for the intelligent identification of important targets in water environmental samples.

Chromosome 8p engineering reveals increased metastatic potential targetable by patient-specific synthetic lethality in liver cancer.

Large-scale chromosomal aberrations are prevalent in human cancer, but their function remains poorly understood. We established chromosome-engineered hepatocellular carcinoma cell lines using CRISPR-Cas9 genome editing. A 33-mega-base pair region on chromosome 8p (chr8p) was heterozygously deleted, mimicking a frequently observed chromosomal deletion. Using this isogenic model system, we delineated the functional consequences of chr8p loss and its impact on metastatic behavior and patient survival. We found that metastasis-associated genes on chr8p act in concert to induce an aggressive and invasive phenotype characteristic for chr8p-deleted tumors. Genome-wide CRISPR-Cas9 viability screening in isogenic chr8p-deleted cells served as a powerful tool to find previously unidentified synthetic lethal targets and vulnerabilities accompanying patient-specific chromosomal alterations. Using this target identification strategy, we showed that chr8p deletion sensitizes tumor cells to targeting of the reactive oxygen sanitizing enzyme Nudix hydrolase 17. Thus, chromosomal engineering allowed for the identification of novel synthetic lethalities specific to chr8p loss of heterozygosity.

TMIC-86. SPATIAL ORGANIZATION OF THE PEDIATRIC HIGH-GRADE GLIOMA TUMOR-IMMUNE LANDSCAPE

Abstract BACKGROUND Advances in cellular immunotherapies have shown promising results for brain tumors, including pediatric-type diffuse high-grade gliomas (pHGG). A comprehensive understanding of the tumor-immune microenvironment (TME) is essential for efficient target identification and novel therapeutic strategies. The interplay between cells and their spatial localization within the TME may affect immune cell phenotype and function. We characterized these interactions and phenotypes by single-cell (SC) spatial proteomic analysis. METHODS Thirty-two tissue biopsies of pHGGs were collected from the Princess Máxima Center biobank consisting of 14 diffuse midline gliomas, H3K27-altered (DMG) and 18 other pHGGs. Three cores per biopsy were assembled in a tissue micro-array (TMA). A cyclical multiplex immunofluorescence antibody staining technique by means of chemical antibody-removal was applied to the TMA sections. This resulted in hyperplexed images enabling detailed mapping of the spatial organization. Survival data of patients was collected retrospectively. Clusters of interacting cells were detected using cellular neighborhood (CN) analysis and SC distance analysis. RESULTS SC analysis revealed a large fraction of myeloid cells in the TME, up to 21% of total cells within a tumor, characterized by expression of IBA1 and CD68. We also identified some significant differences in the composition and spatial organization of DMGs compared to other pHGGs. More specifically, DMG biopsies showed lower abundance of CD163+/CD206+ immunosuppressive myeloid cells compared to other pHGGs (p< 0.001). Additionally, the presence of T cells was significantly lower in DMGs compared to other pHGGs (p< 0.001). CN analysis revealed a higher occurrence of myeloid/astrocyte-like neighborhoods in pHGGs other than DMG. The presence of this cluster in these tumors is associated with higher risk of dying (p< 0.05). Thus, the combination of our spatial proteomic SC technology framework and unique patient sample collection will offer essential insights into the cellular interplay in the TME, and its influence on patient outcomes.

Epigenetic target identification strategy based on multi-feature learning

The identification of potential epigenetic targets for a known bioactive compound is essential and promising as more and more epigenetic drugs are used in cancer clinical treatment and the availability of chemogenomic data related to epigenetics increases. In this study, we introduce a novel epigenetic target identification strategy (ETI-Strategy) that integrates a multi-task graph convolutional neural network prior model and a protein-ligand interaction classification discriminating model using large-scale bioactivity data for a panel of 55 epigenetic targets. Our approach utilizes machine learning techniques to achieve an AUC value of 0.934 for the prior model and 0.830 for the discriminating model, outperforming inverse docking in predicting protein-ligand interactions. When comparing with other open-source target identification tools, it was found that only our tool was able to accurately predict all the targets corresponding to each compound. This further demonstrates the ability of our strategy to take full advantage of molecular-level information as well as protein-level information in molecular activity prediction. Our work highlights the contribution of machine learning in the identification of potential epigenetic targets and offers a novel approach for epigenetic drug discovery and development. Communicated by Ramaswamy H. Sarma

Orthogonal activation of a DNA molecular reaction network for proteinase-free and highly specific mRNA imaging in live cells

Construction of artificial DNA molecular reaction networks (DMRNs) represents an effective approach in biosensing. Here we developed a reaction network consisting of a dual catalytic hairpin assembly-mediated tandem molecular circuit combined with strand displacement reaction (DCHA-SDR) for proteinase-free and highly specific mRNA imaging in living cells. The strand displacement reaction (SDR) was designed to be orthogonally activated in exact order by two fuel strands that separately split into inactive species and grafted into the dual catalytic hairpin assembly-mediated molecular tandem circuit (DCHA). The fuel strands can be activated only when the specific mRNA is introduced, and DCHA is initiated by using two different pieces of mRNA sequences as target. In this way, the target mRNA can be automatically matched twice to obtain an accurate and amplified recognition. Comparative analysis indicates that this orthogonal strategy for target identification is superior to the strategy consisting of single CHA-mediated molecular circuit and strand displacement reaction (SCHA-SDR). Moreover, we demonstrate that the DCHA-SDR system comes with the benefit of proteinase-free progress, which makes the approach suitable for operation in living cells. Final application in various cancer cells and normal cells reveals its enormous potential for highly specific mRNA imaging in biological systems.

Targeted protein degradation reveals BET bromodomains as the cellular target of Hedgehog pathway inhibitor-1

Target deconvolution of small molecule hits from phenotypic screens presents a major challenge. Many screens have been conducted to find inhibitors for the Hedgehog signaling pathway – a developmental pathway with many implications in health and disease – yielding many hits but only few identified cellular targets. We here present a strategy for target identification based on Proteolysis-Targeting Chimeras (PROTACs), combined with label-free quantitative proteomics. We develop a PROTAC based on Hedgehog Pathway Inhibitor-1 (HPI-1), a phenotypic screen hit with unknown cellular target. Using this Hedgehog Pathway PROTAC (HPP) we identify and validate BET bromodomains as the cellular targets of HPI-1. Furthermore, we find that HPP-9 is a long-acting Hedgehog pathway inhibitor through prolonged BET bromodomain degradation. Collectively, we provide a powerful PROTAC-based approach for target deconvolution, that answers the longstanding question of the cellular target of HPI-1 and yields a PROTAC that acts on the Hedgehog pathway.

Identification of RNA-Binding Protein Targets with HyperTRIBE in Saccharomyces cerevisiae.

As a master regulator in cells, RNA-binding protein (RBP) plays critical roles in organismal development, metabolism and various diseases. It regulates gene expression at various levels mostly by specific recognition of target RNA. The traditional CLIP-seq method to detect transcriptome-wide RNA targets of RBP is less efficient in yeast due to the low UV transmissivity of their cell walls. Here, we established an efficient HyperTRIBE (Targets of RNA-binding proteins Identified By Editing) in yeast, by fusing an RBP to the hyper-active catalytic domain of human RNA editing enzyme ADAR2 and expressing the fusion protein in yeast cells. The target transcripts of RBP were marked with new RNA editing events and identified by high-throughput sequencing. We successfully applied HyperTRIBE to identifying the RNA targets of two yeast RBPs, KHD1 and BFR1. The antibody-free HyperTRIBE has competitive advantages including a low background, high sensitivity and reproducibility, as well as a simple library preparation procedure, providing a reliable strategy for RBP target identification in Saccharomyces cerevisiae.

CETSA and thermal proteome profiling strategies for target identification and drug discovery of natural products

BackgroundMonitoring target engagement at various stages of drug development is essential for natural product (NP)-based drug discovery and development. The cellular thermal shift assay (CETSA) developed in 2013 is a novel, broadly applicable, label-free biophysical assay based on the principle of ligand-induced thermal stabilization of target proteins, which enables direct assessment of drug-target engagement in physiologically relevant contexts, including intact cells, cell lysates and tissues. This review aims to provide an overview of the work principles of CETSA and its derivative strategies and their recent progress in protein target validation, target identification and drug lead discovery of NPs. MethodsA literature-based survey was conducted using the Web of Science and PubMed databases. The required information was reviewed and discussed to highlight the important role of CETSA-derived strategies in NP studies. ResultsAfter nearly ten years of upgrading and evolution, CETSA has been mainly developed into three formats: classic Western blotting (WB)-CETSA for target validation, thermal proteome profiling (TPP, also known as MS-CETSA) for unbiased proteome-wide target identification, and high-throughput (HT)-CETSA for drug hit discovery and lead optimization. Importantly, the application possibilities of a variety of TPP approaches for the target discovery of bioactive NPs are highlighted and discussed, including TPP-temperature range (TPP-TR), TPP-compound concentration range (TPP-CCR), two-dimensional TPP (2D-TPP), cell surface-TPP (CS-TPP), simplified TPP (STPP), thermal stability shift-based fluorescence difference in 2D gel electrophoresis (TS-FITGE) and precipitate supported TPP (PSTPP). In addition, the key advantages, limitations and future outlook of CETSA strategies for NP studies are discussed. ConclusionThe accumulation of CETSA-based data can significantly accelerate the elucidation of the mechanism of action and drug lead discovery of NPs, and provide strong evidence for NP treatment against certain diseases. The CETSA strategy will certainly bring a great return far beyond the initial investment and open up more possibilities for future NP-based drug research and development.

Procyanidin B2 3,3″-di-O-gallate suppresses IFN-γ production in murine CD4+ T cells through the regulation of glutamine influx via direct interaction with ASCT2.

Excessive activation of CD4+ T cells increases cytokine production substantially and induces immune-mediated diseases. Procyanidins are polyphenols with anti-inflammatory properties. Procyanidin B2 (PCB2) gallate [specifically, PCB2 3,3''-di-O-gallate (PCB2DG)] inhibits cytokine production through the suppression of glycolysis via mammalian target of rapamycin (mTOR) in T cells. Several amino acids play critical roles in T cell activation, especially glutamine, which is important in mTOR signaling and interferon-γ (IFN-γ) production in CD4+ T cells. However, the mechanisms underlying the effects of PCB2DG, including its interaction partners, have yet to be clarified. In the present study, the mechanisms underlying the inhibitory effect of PCB2DG on IFN-γ through glutamine metabolism regulation were investigated. We found that PCB2DG treatment reduced intracellular glutamine levels in CD4+ T cells, whereas the addition of glutamine abrogated the inhibitory effects of PCB2DG on IFN-γ production. The PCB2DG-induced reduction in intracellular glutamine accumulation led to the upregulated expression of activating transcription factor 4, which was induced by the cytoprotective signaling pathway in the amino acid response. In addition, the mRNA and protein expression levels of alanine serine cysteine transporter 2 (ASCT2), a major glutamine transporter in CD4+ T cells, were not altered by PCB2DG treatment. Further analysis using a target identification strategy revealed that PCB2DG binds to ASCT2, suggesting that PCB2DG interacts directly with this major glutamine transporter to inhibit glutamine influx. Overall, this study indicates that ASCT2 is a novel target protein of a dietary polyphenol and provides new insights into the mechanism underlying the immunomodulatory effects of polyphenols.

Generation of an Aptamer Targeting Receptor-Type Tyrosine-Protein Phosphatase F

Cell-SELEX is a powerful tool to generate aptamers that specifically bind the native molecules on living cells. Here, we report an aptamer ZAJ4a generated by cell-SELEX. The molecular target of ZAJ4a was pulled down by the enriched cell-SELEX pool and identified to be the receptor-type tyrosine-protein phosphatase F (PTPRF) through a stable isotope labeling using amino acids in cell culture (SILAC)-based quantitative proteomic method. ZAJ4a showed high binding affinity with nanomolar range to cancer cells expressing PTPRF. Meanwhile, PTPRF was proven to highly express on several cancer cell lines using ZAJ4a as a molecular probe and to highly express in many kinds of cancer samples using gene expression profiling interactive analysis (GEPIA2) from the TCGA and GTEx databases. These results indicate that the aptamer generated by cell-SELEX showed good specificity at the molecular level. This cell-SELEX and target identification strategies show great potential for identifying biomarkers on the cell surface.

New Wine in an Old Bottle: Utilizing Chemical Genetics to Dissect Apical Hook Development.

The apical hook is formed by dicot seedlings to protect the tender shoot apical meristem during soil emergence. Regulated by many phytohormones, the apical hook has been taken as a model to study the crosstalk between individual signaling pathways. Over recent decades, the roles of different phytohormones and environmental signals in apical hook development have been illustrated. However, key regulators downstream of canonical hormone signaling have rarely been identified via classical genetics screening, possibly due to genetic redundancy and/or lethal mutation. Chemical genetics that utilize small molecules to perturb and elucidate biological processes could provide a complementary strategy to overcome the limitations in classical genetics. In this review, we summarize current progress in hormonal regulation of the apical hook, and previously reported chemical tools that could assist the understanding of this complex developmental process. We also provide insight into novel strategies for chemical screening and target identification, which could possibly lead to discoveries of new regulatory components in apical hook development, or unidentified signaling crosstalk that is overlooked by classical genetics screening.

Identification of A2BAR as a potential target in colorectal cancer using novel fluorescent GPCR ligands

G-protein coupled receptors (GPCRs) have been largely targeted in a wide range of diseases, but few therapies have been directed against GPCRs in the field of cancer, partly because of the lack of effective target identification strategies. Here, using colorectal cancer (CRC) as a model, we explored the gene expression of a panel of GPCRs in tumor and stromal cells, identifying specific gene sets defining each cellular compartment. We selected the adenosine receptor 2B (A2BAR), specifically expressed in cancer cell lines compared with stromal cells, to explore the use of fluorescent ligands that can be used for target visualization. Fluorescent probes allowed semi-quantitative receptor mapping in living cells and validated the specific expression of A2BAR in CRC cell lines. As well, fluorescent ligands were effective at monitoring real-time A2BAR receptor labeling using live-imaging modalities, and displayed high efficiency when used to label complex 3D cellular systems such as tumor spheroids. Finally, we validated A2BAR as a potential pharmacological tool in CRC, using selective antagonists, finding a reduction in tumor cell proliferation. This proof-of-concept study suggests the use of fluorescent ligands for GPCR characterization through imaging, and as possible new tools used for target validation in drug screening methodologies.

A new strategy for the rapid identification and validation of direct toxicity targets of psoralen-induced hepatotoxicity

The interaction between small-molecule compounds of traditional Chinese medicine and their direct targets is the molecular initiation event, which is the key factor for toxicity efficacy. Psoralen, an active component of Fructus Psoraleae, is toxic to the liver and has various pharmacological properties. Although the mechanism of psoralen-induced hepatotoxicity has been studied, the direct target of psoralen remains unclear. Thus, the aim of this study was to discover direct targets of psoralen. To this end, we initially used proteomics based on drug affinity responsive target stability (DARTS) technology to identify the direct targets of psoralen. Next, we used surface plasmon resonance (SPR) analysis and verified the affinity effect of the ‘component-target protein’. This method combines molecular docking technology to explore binding sites between small molecules and proteins. SPR and molecular docking confirmed that psoralen and tyrosine-protein kinase ABL1 could be stably combined. Based on the above experimental results, ABL1 is a potential direct target of psoralen-induced hepatotoxicity. Finally, the targets Nrf2 and mTOR, which are closely related to the hepatotoxicity caused by psoralen, were predicted by integrating proteomics and network pharmacology. The direct target ABL1 is located upstream of Nrf2 and mTOR, Nrf2 can influence the expression of mTOR by affecting the level of reactive oxygen species. Immunofluorescence experiments and western blot results showed that psoralen could affect ROS levels and downstream Nrf2 and mTOR protein changes, whereas the ABL1 inhibitor imatinib and ABL1 agonist DPH could enhance or inhibit this effect. In summary, we speculated that when psoralen causes hepatotoxicity, it acts on the direct target ABL1, resulting in a decrease in Nrf2 expression, an increase in ROS levels and a reduction in mTOR expression, which may cause cell death. We developed a new strategy for predicting and validating the direct targets of psoralen. This strategy identified the toxic target, ABL1, and the potential toxic mechanism of psoralen.

Gold Nanoparticle-Based Photo-Cross-Linking Strategy for Cellular Target Identification of Supercomplex Molecular Systems.

Cellular target identification plays an essential role in innovative drug development and pharmacological mechanism elucidation. However, very few practical experimental methodologies have been developed for identifying target proteins for supercomplex molecular systems such as biologically active phytochemicals or pharmaceutical compositions. To overcome this limitation, we synthesized gold nanoparticles (AuNPs) as solid scaffolds, which were bound with 4,4'-dihydroxybenzophenone (DHBP) as a photo-cross-linking group on the surface. Then, DHBP-modified AuNPs cross-linked various organic compounds from phytochemicals under ultraviolet radiation via carbene reactions, H-C bond insertion, for catalytic C-C bond formation. We next used the phytochemical-cross-linked AuNPs (phytoAuNPs) to pull down potential binding proteins from brain tissue lysate and identified 13 neuroprotective targets by mass spectrometry analysis. As an exemplary study, we selected Hsp60 as a crucial cellular target to further screen 14 target-binding compounds from phytochemicals through surface plasmon resonance (SPR) analysis, followed by Hsp60 activity detection and neuroprotective effect assay in cells. Collectively, this gold nanoparticle-based photo-cross-linking strategy can serve as a useful platform for discovering novel cellular targets for supercomplex molecular systems and help to explore pharmacological mechanisms and active substances.

A New Strategy for the Rapid Identification and Validation of the Direct Targets of Aconitine-Induced Cardiotoxicity.

BackgroundThe interaction of small molecules with direct targets constitutes the molecular initiation events of drug efficacy and toxicity. Aconitine, an active compound of the Aconitum species, has various pharmacological effects but is strongly toxic to the heart. The direct targets of aconitine-induced cardiotoxicity remain unclear.MethodsWe predicted the toxic targets of aconitine based on network pharmacology and followed a novel proteomic approach based on the “drug affinity responsive target stability” technology combined with LC-MS/MS to identify the direct targets of aconitine. The identified targets were analysed from the perspective of multilevel and multidimensional bioinformatics through a network integration method. The binding sites were investigated via molecular docking to explore the toxicity mechanism and predict the direct targets of aconitine. Finally, atomic force microscopy (AFM) imaging was performed to verify the affinity of aconitine to the direct targets.ResultsPTGS2, predicted by network pharmacology as a toxic target, encodes cyclooxygenase 2 (COX-2), which is closely related to myocardial injury. Furthermore, cytosolic phospholipase A2 (cPLA2) is the upstream signal protein of PTGS2, and it is a key enzyme in the metabolism of arachidonic acid during an inflammatory response. We determined cPLA2 as a direct target, and AFM imaging verified that aconitine could bind to cPLA2 well; thus, aconitine may cause the expression of PTGS2/COX-2 and release inflammatory factors, thereby promoting myocardial injury and dysfunction.ConclusionWe developed a complete set of methods to predict and verify the direct targets of aconitine, and cPLA2 was identified as one. Overall, the novel strategy provides new insights into the discovery of direct targets and the molecular mechanism of toxic components that are found in traditional Chinese medicine.

In This Issue

See article, p. 568.Hematologic malignancy patients are highly susceptible to severe coronavirus disease-19 (COVID-19) illness and are a high-priority group for vaccination to mitigate COVID-19–related morbidity and mortality. Chung et al. find differential COVID-19 vaccine humoral responses between hematologic malignancy subtypes and disease-specific therapies across patient cohorts. Hematologic malignancy patients undergoing active therapy have baseline humoral responses that are further suppressed. Importantly, antibody titers alone are an imperfect measure of immunity, as many patients with positive anti–SARS-CoV-2 antibody titers have insufficient neutralizing capacity. These findings together provide important metrics for optimizing the clinical care of this vulnerable population.See article, p. 577.Cellular therapies, including allogeneic and autologous hematopoietic cell transplant (allo-HCT, auto-HCT) and chimeric antigen receptor T-cell therapy (CAR T), render patients severely immunocompromised for extended periods after therapy. Tamari et al. define predictors of response to COVID-19 vaccine to nominate important factors for optimizing response to vaccination. Type of cellular therapy, timing of vaccination post–cellular therapy, and immune recovery post–cellular therapy predict response. Many patients with positive, yet low, anti–SARS-CoV-2 antibody titers demonstrate low neutralizing capacity, highlighting the limitations of using antibody titer as the only proxy for immunity.See article, p. 586.Pediatric acute leukemias have historically been classified by immunophenotyping followed by genomic subclassification. Patients are categorized into T, B, myeloid, or mixed-lineage disease for the purpose of general treatment approach, and these regimens are then further tailored in intensity and/or targeted therapeutics when applicable. Fornerod et al. apply an integrated analysis of gene expression and mutation profiling to a cohort of pediatric patients with acute leukemias that have myeloid features. This reveals a subset of patients with shared mutational composition and gene expression that spanned three immunophenotypes: T, myeloid, and mixed-lineage acute leukemias. The authors further identify patients with the same mutations but with differential gene expression profiles and outcomes. These results suggest acute leukemia classification requires more comprehensive, sequencing-based diagnostic methods beyond flow cytometry.See article, p. 600.Waldenstrom macroglobulinemia (WM) is characterized by outgrowth of mature IgM+ B cells with MYD88 mutations. Kaushal et al. show that WM lesions originate in the presence of several alterations in the immune microenvironment, including aberrant nonmalignant extrafollicular B-cell expansion, myeloid inflammation, and WM-specific T-cell immune responses. The characteristic MyD88 mutations originate in early lymphoid progenitors prior to expansion of the malignant clone. These findings suggest a new model for the origin of WM.See article, p. 616.Overall outcomes are dismal in relapsed/refractory AML (R/R AML), and therapeutic options are limited. In this phase II study, Zeidner et al. investigate the safety, feasibility, and clinical activity of immune checkpoint blockade with pembrolizumab after high-dose cytarabine (HiDAC) chemotherapy in R/R AML. The addition of pembrolizumab to HiDAC leads to an overall complete remission rate of 38%, with encouraging clinical outcomes and self-limiting immune-related adverse events. Multifaceted immunogenomic profiling before and after therapy reveals putative biomarkers of response to pembrolizumab, warranting further investigation.See article, p. 630.Inhibitors of Bruton tyrosine kinase (BTKi) block pathogenic B-cell receptor (BCR) signaling, improving outcomes for patients with lymphoid malignancies. Nonetheless, resistance to BTK therapy is a frequent clinical problem that has previously been ascribed to genetic alterations. Shaffer et al. find a primary, early mechanism of BTKi resistance involving epigenetic reprogramming of the malignant cell. Specifically, the epigenetic upregulation of the small GTPase RAC2 increases its interaction with, and activation of, PLCγ2. This RAC2-dependent activation circumvents the requirement for BTK to phosphorylate PLCγ2 to trigger the prosurvival NF-κB pathway. Clinically available drugs are identified that can kill BTKi-resistant cells.See article, p. 648.Clinical translation of CAR T-cell therapy for AML has been limited by the lack of optimal CAR targets and the risk of on-target, off-tumor toxicity. Richards et al. report a novel anti-AML CAR T-cell therapy targeting CD93 and demonstrate antileukemic activity without toxicity to critical hematopoietic stem and progenitor cells. To mitigate tissue-specificity concerns, next-generation, NOT-gated CD93 CAR T cells were engineered. Transcriptomic analysis of AML and endothelial cells demonstrates a promising target identification strategy for further advancement of this technology.

Currently Available Strategies for Target Identification of Bioactive Natural Products.

In recent years, biologically active natural products have gradually become important agents in the field of drug research and development because of their wide availability and variety. However, the target sites of many natural products are yet to be identified, which is a setback in the pharmaceutical industry and has seriously hindered the translation of research findings of these natural products as viable candidates for new drug exploitation. This review systematically describes the commonly used strategies for target identification via the application of probe and non-probe approaches. The merits and demerits of each method were summarized using recent examples, with the goal of comparing currently available methods and selecting the optimum techniques for identifying the targets of bioactive natural products.