Target proteins-regulated DNA nanomachine-electroactive substance complexes enable separation-free electrochemical detection of clinical exosome.

Simple and reliable profiling of tumor-derived exosomes (TDEs) holds significant promise for the early detection of cancer. Nonetheless, this remains challenging owing to the substantial heterogeneity and low concentration of TDEs. Herein, we devised an accurate and highly sensitive electrochemical sensing strategy for TDEs via simultaneously targeting exosomal mucin 1 (MUC1) and programmed cell death ligand 1 (PD-L1). This approach employs high-affinity aptamers as specific recognition elements, utilizes rolling circle amplification and DNA nanospheres as effective bridges and signal amplifiers, and leverages methylene blue (MB) and doxorubicin (DOX) as robust signal reporters. The crux of this separation- and label-free method is the specific response of MB and DOX to G-quadruplex structures and DNA nanospheres, respectively. Quantifying TDEs using this strategy enabled precise discrimination of lung cancer patients (n = 25) from healthy donors (n = 12), showing 100% specificity (12/12), 92% sensitivity (23/25), and an overall accuracy of 94.6% (35/37), with an area under the receiver operating characteristic curve (AUC) of 0.97. Furthermore, the assay results strongly correlated with findings from computerized tomography and pathological analyses. Our approach could facilitate the early diagnosis of lung cancer through TDEs-based liquid biopsy.

Current advances and development strategies of targeting son of sevenless 1 (SOS1) in drug discovery.

The Son of Sevenless 1 (SOS1) guanine nucleotide exchange factor, prevalent across eukaryotic species, plays a pivotal role in facilitating the attachment of RAS protein to GTP, thereby regulating the activation of intracellular RAS proteins. This regulation is part of a feedback mechanism involving SOS1, which allows both activators and inhibitors of SOS1 to exert control over downstream signaling pathways, demonstrating potential anti-tumor effects. Predominantly, small molecule modulators that target SOS1 focus on a hydrophobic pocket within the CDC25 protein domain. The effectiveness of these modulators largely depends on their ability to interact with specific amino acids, notably Phe890 and Tyr884. This interaction is crucial for influencing the protein-protein interaction (PPI) between RAS and the catalytic domain of SOS1. Currently, most small molecule modulators targeting SOS1 are in the preclinical research phase, with a few advancing to clinical trials. This progression raises safety concerns, making the assurance of drug safety a primary consideration alongside the enhancement of efficacy in the development of SOS1 modulators. This review encapsulates recent advancements in the chemical categorization of SOS1 inhibitors and activators. It delves into the evolution of small molecule modulation targeting SOS1 and offers perspectives on the design of future generations of selective SOS1 small molecule modulators.

The relationship between long non-coding gene CASC21 polymorphisms and cervical cancer.

BACKGROUND: CASC21 was reported to be a hotspot gene in cervical cancer. The relationship between CASC21 genetic polymorphisms and cervical cancer has not been reported. Genetic factors influence the occurrence of cervical cancer. Thus, we explored the correlation between CASC21 polymorphisms and cervical cancer. METHODS: A total of 973 participants within 494 cervical cancer cases and 479 healthy controls were recruited. Five single nucleotide polymorphisms (SNPs) in the CASC21 gene were genotyped using the Agena MassARRAY platform. Chi-squared test, logistic regression analysis, odds ratio (OR), multifactor dimensionality reduction (MDR), and 95% confidence interval (95%CI) were used for data analysis. RESULTS: In the overall analysis, rs16902094 (p = .014, OR = 1.86, 95% CI = 1.12-3.08) and rs16902104 (p = .014, OR = 1.86, 95% CI = 1.12-3.09) had the risk-increasing correlation with the occurrence of cervical cancer. Stratification analysis showed that rs16902094 and rs16902104 were still associated with cervical cancer risk in the subgroups with age > 51, BMI < 24 kg/m2, smokers, and patients with cervical squamous cell carcinoma. MDR analysis displayed that rs16902094 (.49%) and rs16902104 (.52%) were the main influential attribution factor for cervical cancer risk. CONCLUSION: Our finding firstly determined that two CASC21 SNPs (rs16902094, rs16902104) were associated with an increased risk of cervical cancer, which adds to our knowledge regarding the effect of CASC21 on cervical carcinogenesis.

Design, Synthesis, and Biological Evaluation of Dual Inhibitors of EGFRL858R/T790M/ACK1 to Overcome Osimertinib Resistance in Nonsmall Cell Lung Cancers.

Activation of the alternative pathways and abnormal signaling transduction are frequently observed in third-generation EGFR-TKIs (epidermal growth factor receptor tyrosine kinase inhibitors)-resistant patients. Wherein, hyperphosphorylation of ACK1 contributes to EGFR-TKIs acquired resistance. Dual inhibition of EGFRL858R/T790M and ACK1 might improve therapeutic efficacy and overcome resistance in lung cancers treatment. Here, we identified a EGFRL858R/T790M/ACK1 dual-targeting compound 21a with aminoquinazoline scaffold, which showed excellent inhibitory activities against EGFRL858R/T790M (IC50 = 23 nM) and ACK1 (IC50 = 263 nM). The cocrystal and docking analysis showed that 21a occupied the ATP binding pockets of EGFRL858R/T790M and ACK1. Moreover, 21a showed potent antiproliferative activities against the H1975 cells, MCF-7 cells and osimertinib-resistant cells AZDR. Further, 21a showed significant antitumor effects and good safety in ADZR xenograft-bearing mice. Taken together, 21a was a potent dual inhibitor of EGFRL858R/T790M/ACK1, which is deserved as a potential lead for overcoming acquired resistance to osimertinib during the EGFR-targeted therapy.

Single-Cell Liquid Biopsy of Lung Cancer: Ultra-Simplified Efficient Enrichment of Circulating Tumor Cells and Hand-Held Fluorometer Portable Testing.

Herein, we propose a paper-based laboratory via enzyme-free nucleic acid amplification and nanomaterial-assisted cation exchange reactions (CERs) assisted single-cell-level analysis (PLACS). This method allowed for the rapid detection of mucin 1 and trace circulating tumor cells (CTCs) in the peripheral blood of lung cancer patients. Initially, an independently developed method requiring one centrifuge, two reagents (lymphocyte separation solution and erythrocyte lysate), and a three-step, 45 min sample pretreatment was employed. The core of the detection approach consisted of two competitive selective identifications: copper sulfide nanoparticles (CuS NPs) to C-Ag+-C and Ag+, and dual quantum dots (QDs) to Cu2+ and CuS NPs. To facilitate multimodal point-of-care testing (POCT), we integrated solution visualization, test strip length reading, and a self-developed hand-held fluorometer readout. These methods were detectable down to ag/mL of mucin 1 concentration and the single-cell level. Forty-seven clinical samples were assayed by fluorometer, yielding 94% (30/32) sensitivity and 100% (15/15) specificity with an area under the curve (AUC) of 0.945. Nine and 15 samples were retested by a test strip and hand-held fluorometer, respectively, with an AUC of 0.95. All test results were consistent with the clinical imaging and the folate receptor (FR)-PCR kit findings, supporting its potential in early diagnosis and postoperative monitoring.

Biomolecules-mediated electrochemical signals of Cu2+: Y-DNA nanomachines enable homogeneous rapid one-step assay of lung cancer circulating tumor cells.

This study presents a straightforward efficient technique for extracting circulating tumor cells (CTCs) and a rapid one-step electrochemical method (45 min) for detecting lung cancer A549 cells based on the specific recognition of mucin 1 using aptamers and the modulation of Cu2+ electrochemical signals by biomolecules. The CTCs separation and enrichment process can be completed within 45 min using lymphocyte separation solution (LSS), erythrocyte lysis solution (ELS), and three centrifugations. Besides, the influence of various biomolecules on Cu2+ electrochemical signals is comprehensively discussed, with DNA nanospheres selected as the medium. Three single-stranded DNA sequences were hybridized to form Y-shaped DNA (Y-DNA), creating DNA nanospheres. Upon specific capture of mucin 1 by the aptamer, most DNA nanospheres could form complexes with Cu2+ (DNA nanosphere-Cu2+), significantly reducing the concentration of free Cu2+. Our approach yielded the limit of detection (LOD) of 2 ag/mL for mucin 1 and 1 cell/mL for A549 cells. 39 clinical blood samples were used for further validation, yielding results closely correlated with pathological, computed tomography (CT) scan findings and folate receptor-polymerase chain reaction (FR-PCR) kits. The receiver operating characteristic (ROC) curve displayed an area under the curve (AUC) value of 0.960, demonstrating 100% specificity and 93.1% sensitivity for the assay. Taken together, our findings indicate that this straightforward and efficient pretreatment and rapid, highly sensitive electrochemical assay holds great promise for liquid biopsy-based tumor detection using CTCs.

Inhibition of γ-glutamyl transferase suppresses airway hyperresponsiveness and airway inflammation in a mouse model of steroid resistant asthma exacerbation.

Introduction: Despite recent advances, there are limited treatments available for acute asthma exacerbations. Here, we investigated the therapeutic potential of GGsTop, a γ-glutamyl transferase inhibitor, on the disease with a murine model of asthma exacerbation. Methods: GGsTop was administered to mice that received lipopolysaccharide (LPS) and ovalbumin (OVA) challenges. Airway hyperresponsiveness (AHR), lung histology, mucus hypersecretion, and collagen deposition were analyzed to evaluate the hallmark features of asthma exacerbation. The level of proinflammatory cytokines and glutathione were determined with/without GGsTop. The transcription profiles were also examined. Results: GGsTop attenuates hallmark features of the disease with a murine model of LPS and OVA driven asthma exacerbation. Airway hyperresponsiveness (AHR), mucus hypersecretion, collagen deposition, and expression of inflammatory cytokines were dramatically inhibited by GGsTop treatment. Additionally, GGsTop restored the level of glutathione. Using RNA-sequencing and pathway analysis, we demonstrated that the activation of LPS/NFκB signaling pathway in airway was downregulated by GGsTop. Interestingly, further analysis revealed that GGsTop significantly inhibited not only IFNγ responses but also the expression of glucocorticoid-associated molecules, implicating that GGsTop profoundly attenuates inflammatory pathways. Conclusions: Our study suggests that GGsTop is a viable treatment for asthma exacerbation by broadly inhibiting the activation of multiple inflammatory pathways.

X-ray Crystal Structure-Guided Discovery of Novel Indole Analogues as Colchicine-Binding Site Tubulin Inhibitors with Immune-Potentiating and Antitumor Effects against Melanoma.

A series of novel indole analogues were discovered as colchicine-binding site inhibitors of tubulin. Among them, 3a exhibited the highest antiproliferative activity (average IC50 = 4.5 nM), better than colchicine (IC50 = 65.3 nM). The crystal structure of 3a in complex with tubulin was solved by X-ray crystallography, which explained the improved binding affinity of 3a to tubulin and thus its higher anticancer activity (IC50 = 4.5 nM) than the lead compound 12b (IC50 = 32.5 nM). In vivo, 3a (5 mg/kg) displayed significant antitumor efficacy against B16-F10 melanoma with a TGI of 62.96% and enhanced the antitumor efficacy of a small-molecule PD-1/PD-L1 inhibitor NP19 (TGI = 77.85%). Moreover, 3a potentiated the antitumor immunity of NP19 by activating the tumor immune microenvironment, as demonstrated by the increased tumor-infiltrating lymphocytes (TIL). Collectively, this work shows a successful example of crystal structure-guided discovery of a novel tubulin inhibitor 3a as a potential anticancer and immune-potentiating agent.

Design, Synthesis, and Antitumor Activity of Potent and Selective EGFR L858R/T790M Inhibitors and Identification of a Combination Therapy to Overcome Acquired Resistance in Models of Non-small-cell Lung Cancer.

Epidermal growth factor receptor (EGFR) is one of the most studied drug targets for the treatment of non-small-cell lung cancer (NSCLC). Here, we report the identification, structure optimization, and structure-activity relationship studies of quinazoline derivatives as novel selective EGFR L858R/T790M inhibitors. The most promising compound, 28f, exhibited strong inhibitory activity against EGFR L858R/T790M (IC50 = 3.5 nM) and greater than 368-fold selectivity over EGFR WT (IC50 = 1290 nM), a 6.7-fold improvement over osimertinib. Furthermore, 28f effectively inhibited downstream signaling pathways and induced apoptosis in mutant cells. In the H1975 xenograft in vivo model, 28f exhibited a good tumor suppressive effect. Furthermore, the combination of 28f with the ACK1 inhibitor dasatinib produced synergistic antiproliferative efficacy with 28f in 28f-resistant cells and in vivo. In conclusion,28f could become a candidate drug for the treatment of NSCLC, and the combination of 28f and dasatinib is expected to overcome EGFR resistance.

Long noncoding RNAs with peptide-encoding potential identified in esophageal squamous cell carcinoma: KDM4A-AS1-encoded peptide weakens cancer cell viability and migratory capacity.

Currently, the knowledge of long noncoding RNA (lncRNA)-encoded peptides is quite lacking in esophageal squamous cell carcinoma (ESCC). In this study, we simultaneously identified six lncRNA open reading frames (ORFs) with peptide-coding abilities including lysine-specific demethylase 4A antisense RNA 1 (KDM4A-AS1) ORF by combining weighted gene co-expression network analysis (WGCNA) for ESCC clinical samples, ribosome footprints, ORF prediction, mass spectrometry (MS) identification, and western blotting. KDM4A-AS1 ORF-encoded peptide reduced ESCC cell viability and migratory ability. Co-immunoprecipitation and MS analysis revealed that KDM4A-AS1-encoded peptide specifically bound with 103 proteins in ESCC cells, and enrichment analysis suggested that peptide-bound proteins were related to fatty acid metabolism and redox process. Cell and molecular experiments demonstrated that KDM4A-AS1-encoded peptide inhibited stearoyl-CoA desaturase and fatty acid synthase expression, increased reactive oxygen species level, and reduced mitochondrial membrane potential in ESCC cells. In summary, multiple lncRNAs with translation potential were simultaneously identified by combining multiple approaches in ESCC, providing novel identification strategies for lncRNA-encoded peptides. Moreover, lncRNA KDM4A-AS1-encoded peptide weakened ESCC cell viability and migratory capacity and functioned in fatty acid metabolism and redox process.

RSK inhibitors as potential anticancer agents: Discovery, optimization, and challenges.

Ribosomal S6 kinase (RSK) family is a group of serine/threonine kinases, including four isoforms (RSK1/2/3/4). As a downstream effector of the Ras-mitogen-activated protein kinase (Ras-MAPK) pathway, RSK participates in many physiological activities such as cell growth, proliferation, and migration, and is intimately involved in tumor occurrence and development. As a result, it is recognized as a potential target for anti-cancer and anti-resistance therapies. There have been several RSK inhibitors discovered or designed in recent decades, but only two have entered clinical trials. Low specificity, low selectivity, and poor pharmacokinetic properties in vivo limit their clinical translation. Published studies performed structure optimization by increasing interaction with RSK, avoiding hydrolysis of pharmacophores, eliminating chirality, adapting to binding site shape, and becoming prodrugs. Besides enhancing efficacy, the focus of further design will move towards selectivity since there are functional differences among RSK isoforms. This review summarized the types of cancers associated with RSK, along with the structural characteristics and optimization process of the reported RSK inhibitors. Furthermore, we addressed the importance of RSK inhibitors' selectivity and discussed future drug development directions. This review is expected to shed light on the emergence of RSK inhibitors with high potency, specificity, and selectivity.

Design, Synthesis, and Biological Evaluation of Heterocyclic-Fused Pyrimidine Chemotypes Guided by X-ray Crystal Structure with Potential Antitumor and Anti-multidrug Resistance Efficacy Targeting the Colchicine Binding Site.

Herein, a series of quinazoline and heterocyclic fused pyrimidine analogues were designed and synthesized based on the X-ray co-crystal structure of lead compound 3a, showing efficacious antitumor activities. Two analogues, 15 and 27a, exhibited favorable antiproliferative activities, which were more potent than lead compound 3a by 10-fold in MCF-7 cells. In addition, 15 and 27a exhibited potent antitumor efficacy and tubulin polymerization inhibition in vitro. 15 reduced the average tumor volume by 80.30% (2 mg/kg) in the MCF-7 xenograft model and 75.36% (4 mg/kg) in the A2780/T xenograft model, respectively. Most importantly, supported by structural optimization and Mulliken charge calculation, X-ray co-crystal structures of compounds 15, 27a, and 27b in complex with tubulin were resolved. In summary, our research provided the rational design strategy of colchicine binding site inhibitors (CBSIs) based on X-ray crystallography with antiproliferation, antiangiogenesis, and anti-multidrug resistance properties.

Discovery and biological evaluation of 4,6-pyrimidine analogues with potential anticancer agents as novel colchicine binding site inhibitors.

Novel 4,6-pyrimidine analogues were designed and synthesized as colchicine binding site inhibitors (CBSIs) with potent antiproliferative activities. Among them, compound 17j has the most potent activities against 6 human cancer cell lines with IC50 values from 1.1 nM to 4.4 nM, which was 76 times higher than the lead compound 3 in A549 cells. The co-crystal structure of 17j in complex with tubulin confirms the key binding mode at the colchicine binding site. Moreover, 17j inhibited the tubulin polymerization in biochemical assays, depolymerized cellular microtubules, induced the G2/M arrest, inhibited the cell migration, and promoted the initiation of apoptosis. In vivo, 17j effectively inhibits primary tumor growth with tumor growth inhibition rates of 42.51% (5 mg/kg) and 65.42% (10 mg/kg) in A549 xenograft model. Taken together, 17j represents a promising new generation of CBSIs.

Generative deep learning enables the discovery of a potent and selective RIPK1 inhibitor.

The retrieval of hit/lead compounds with novel scaffolds during early drug development is an important but challenging task. Various generative models have been proposed to create drug-like molecules. However, the capacity of these generative models to design wet-lab-validated and target-specific molecules with novel scaffolds has hardly been verified. We herein propose a generative deep learning (GDL) model, a distribution-learning conditional recurrent neural network (cRNN), to generate tailor-made virtual compound libraries for given biological targets. The GDL model is then applied to RIPK1. Virtual screening against the generated tailor-made compound library and subsequent bioactivity evaluation lead to the discovery of a potent and selective RIPK1 inhibitor with a previously unreported scaffold, RI-962. This compound displays potent in vitro activity in protecting cells from necroptosis, and good in vivo efficacy in two inflammatory models. Collectively, the findings prove the capacity of our GDL model in generating hit/lead compounds with unreported scaffolds, highlighting a great potential of deep learning in drug discovery.

The Low Endometrial Expression of Long Non-Coding RNA NORAD Is Associated with Recurrent Pregnancy Losses and Unexplained Infertility.

Objective: Unexplained infertility (UIF) or recurrent pregnancy loss (RPL) affects 10%-15% of couples in their reproductive years and is multifactorial and not completely elucidated. In this study, we attempt to determine the endometrial expression pattern of non-coding RNA activated by DNA damage (NORAD) in women with UIF and RPL, as well as its clinical significance. Methods: The microarray dataset GSE165004 was used to identify differentially expressed RNAs in the endometrial samples between women with RPL and fertile women and between women with UIF and fertile women. A total of 142 women were included in this retrospective analysis, including 32 women with UIF, 48 women with RPL, and 62 fertile women. The relative expression level of NORAD in the endometrial tissues was quantified by qRT-PCR. Results: NORAD stood out as an only overlapped lncRNA among differentially expressed RNAs in the endometrial samples between RPL and fertile women and between UIF and fertile women. It was showed that the endometrial tissues of UIF and RPL both were demonstrated with lower relative expression levels of NORAD (UIF: 2.09 ± 0.68; RPL: 1.98 ± 0.65) than the endometrial tissues of normal fertility (4.32 ± 1.04) (P < 0.001). Pearson correlation analysis demonstrated that the serum level of E2 was negatively correlated with the relative expression level of NORAD in the endometrial tissues of UIF (r = -0.630) and RPL (r = -0.696). Results of ROC curves showed that the endometrial expression of NORAD could be used to differentiate RPL and UIF with an AUC of 0.977 (95% CI: 0.956-0.999) and 0.970 (95% CI: 0.941-0.998), sensitivity of 0.873 and 0.955, and specificity of 0.845 and 0.948, respectively. Conclusion: The findings obtained from the study showed that the low endometrial expression of NORAD was linked to fertility-related problems, such as UIF and RPL.

A paper-based assay for the colorimetric detection of SARS-CoV-2 variants at single-nucleotide resolution.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for versatile diagnostic assays that can discriminate among emerging variants of the virus. Here we report the development and performance benchmarking of an inexpensive (approximately US$0.30 per test) assay for the rapid (sample-to-answer time within 30 min) colorimetric detection of SARS-CoV-2 variants. The assay, which we integrated into foldable paper strips, leverages nucleic acid strand-displacement reactions, the thermodynamic energy penalty associated with single-base-pair mismatches and the metal-ion-controlled enzymatic cleavage of urea to amplify the recognition of viral RNAs for the colorimetric readout of changes in pH via a smartphone. For 50 throat swab samples, the assay simultaneously detected the presence of SARS-CoV-2 and mutations specific to the SARS-CoV-2 variants Alpha, Beta and Gamma, with 100% concordance with real-time quantitative polymerase chain reaction and RNA sequencing. Customizable and inexpensive paper-based assays for the detection of viruses and their variants may facilitate viral surveillance.

DCLK1 autoinhibition and activation in tumorigenesis.

Doublecortin-like kinase 1 (DCLK1) is upregulated in many tumors and is a marker for tumor stem cells. Accumulating evidence suggests DCLK1 constitutes a promising drug target for cancer therapy. However, the regulation of DCLK1 kinase activity is poorly understood, particularly the function of its autoinhibitory domain (AID), and, moreover, no physiological activators of DCLK1 have presently been reported. Here we determined the first DCLK1 kinase structure in the autoinhibited state and identified the neuronal calcium sensor HPCAL1 as an activator of DCLK1. The C-terminal AID functions to block the ATP-binding site and is competitive with ATP. HPCAL1 binds directly to the AID in a Ca2+-dependent manner, which releases the autoinhibition. We also analyzed cancer-associated mutations occurring in the AID and elucidate how these mutations disrupt DCLK1 autoinhibition to elicit kinase activity upregulation. Our results present a molecular mechanism for autoinhibition and activation of DCLK1 kinase activity and provide insights into DCLK1-associated tumorigenesis.

Isothermal RNA Amplification for the Detection of Viable Pathogenic Bacteria to Estimate the Salmonella Virulence for Causing Enteritis.

Viable foodborne pathogens can cause intestinal infection and food poisoning. Herein, we reported an RNA assay allowing for sensitive (close to 1 CFU and 1% viable bacteria detectable) and rapid (within 2.5 h) detection of viable pathogenic bacteria by coupling isothermal RNA amplification (nucleic acid sequence-based amplification, NASBA) with a CRISPR/Cas13a system. NASBA allowed direct amplification of 16S rRNA extracted from viable S. enterica (RNAs degrade rapidly in dead bacteria), and the specificity of amplification was ensured using Cas13a/crRNA to recognize the amplicons. We used the CRISPR/Cas13-based NASBA assay (termed cNASBA assay) to investigate the in vivo colonization and intestinal infection of S. enterica in mice. We found that S. enterica was mainly colonized at the cecum, colon, and rectum, and the severity of enteritis caused by S. enterica was determined by the number of viable S. enterica rather than the total count of S. enterica. The cNASBA assay can quantify viable S. enterica and thus can improve the accuracy of virulence estimation compared to qPCR. It shows promise as a reliable tool for monitoring pathogen contamination and biosafety control.

A distinct structure of Cas1-Cas2 complex provides insights into the mechanism for the longer spacer acquisition in Pyrococcus furiosus.

In the adaptation stage of CRISPR-Cas systems, the Cas1-Cas2 integrase captures and integrates new invader-derived spacers into the CRISPR locus, serving as a molecular memory of prior infection. As of yet, the structural information of Cas1-Cas2 complex is available only for two species. Here we present the crystal structure of Cas1-Cas2 complex of Pyrococcus furiosus, which showed a distinct architecture from the known Cas1-Cas2 complexes. The shorter C-terminal tail of Pfu Cas2 directs the Cas1 dimers go in the opposite direction, resulting in a different prespacer binding mode. Based on our structural and mutagenesis results, we modeled a prespacer with a shorter duplex and longer 3' overhangs to bind Pfu Cas1-Cas2 complex. The prespacer preference was confirmed by EMSA, fluorescence polarization, and in vitro integration assays. This model provides a potential explanation for the longer spacer acquisition observed in P. furiosus when deleting both cas4 genes. Our study highlights the diversity of the CRISPR adaptation module.