Target Binding Research Articles

Improving Aptamer Affinity and Determining Sequence-Activity Relationships via Motif-SELEX.

The affinity of nucleic acid aptamers isolated in vitro via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is often limited because the entire potential sequence space cannot be screened. In this study, we introduce Motif-SELEX, a novel method that enables the optimization of existing underperforming aptamers by generating libraries that broadly represent both the sequence and length variations of the parent sequence. This approach enables the isolation of sequences with improved affinity without the biases and limitations of traditional mutagenesis methods like doped SELEX and error-prone PCR. As a demonstration, we applied Motif-SELEX to a DNA-based morphine aptamer and a 2' fluoro- and methoxy-RNA-based apixaban aptamer, discovering new, better-performing sequences with differing random domain lengths from their parents and up to 10-fold improvements in affinity. These new sequences would be inaccessible to traditional post-SELEX methods. Critically, our analysis of Motif-SELEX pools also enabled us to identify sequence and structural elements crucial for target binding and to predict secondary and tertiary structures for a given aptamer family─even when those structures involve noncanonical nucleotide interactions. We believe that Motif-SELEX offers an effective and generalizable solution for optimizing the structure and binding properties of functional nucleic acid molecules for diverse applications.

Comparative Structure Activity Relationship Characterization of the Biosimilar BAT1806/BIIB800 to Reference Tocilizumab.

BAT1806/BIIB800 (Tofidence™/tocilizumab-bavi), a biosimilar of tocilizumab, demonstrated a high degree of analytical and functional similarity to reference tocilizumab (TCZ) in a comprehensive comparative analytical assessment. Minor differences with respect to TCZ were observed for some attributes and this study assessed the potential impact of these differences through structure activity relationship characterization. Structure activity relationship studies were conducted to assess glycation, glycosylation, charge variants, hydrophobicity, oxidation, and deamidation differences, using a range of investigative techniques. Structure activity relationship studies were performed on one lot each of BAT1806/BIIB800 and TCZ (European Union sourced only) except for glycation, where additional lots sourced from China and the USA were used. Average total glycated protein content of BAT1806/BIIB800 was higher than TCZ (10.08% vs 1.19%); however, biological activity, including target binding and functional potency, was unaffected. Stress-induced glycation of BAT1806/BIIB800 and TCZ also did not affect the biological activity of the products despite up to 60% total glycation content. Minor differences were observed between BAT1806/BIIB800 and TCZ in glycosylation, charge variants, hydrophobicity, oxidation, and deamidation without a relevant impact on interleukin-6 receptor binding, Fc-receptor binding, and effector functions. In forced degradation studies, oxidation and deamidation trends were comparable between the two products. Comparative structure activity relationship characterization of BAT1806/BIIB800 and TCZ indicated that there are no relevant differences in quality attributes between BAT1806/BIIB800 and reference TCZ. Observed differences between BAT1806/BIIB800 and TCZ had no functional impact on BAT1806/BIIB800. The results support the conclusion that BAT1806/BIIB800 is similar to TCZ.

LINC00968 accelerates osteogenic differentiation of bone marrow mesenchymal stem cells via the miR-17-5p/STAT3 axis.

BMSCs with robust osteogenic differentiation capacity can participate in the repair of osteoporotic (OP) bone. Long non-coding RNAs (LncRNAs) serve as crucial regulators of osteogenic differentiation. This study aims to investigate the clinical implications of LINC00968 in OP and elucidate its molecular mechanisms. Patients with OP and controls without OP were enrolled. RT-qPCR was utilized the quantify the levels of LINC00968, miR-17-5p, STAT3, and osteogenic differentiation markers. ROC curve was conducted to evaluate the diagnostic significance. Osteogenic differentiation medium (OM) induced hBMSCs. Flow cytometry was used to examine apoptosis. DLR and RIP assay were performed to validate target binding. LINC00968 was notably decreased in the serum and bone tissue of patients with OP, whereas it was markedly elevated during osteogenic differentiation of hBMSCs. LINC00968 has 78.65% sensitivity and 71.95% specificity in identifying OP patients from controls. Silencing of LINC00968 sharply diminished ALP activity and osteogenic differentiation markers levels while promoting apoptosis in hBMSCs under OM induction. However, this reduction was notably reversed by the administration of a miR-17-5p inhibitor. Molecularly, miR-17-5p directly targets LINC00968 and STAT3. Our results indicate that LINC00968 downregulation is a diagnostic biomarker for OP, facilitating osteogenic differentiation and inhibiting apoptosis via miR-17-5p/STAT3 axis, suggesting a new therapeutic target for OP progression.

Recent Advances in Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Proteins System-Based Biosensors

High-sensitivity and high-specificity biodetection is critical for advancing applications in life sciences, biosafety, food safety, and environmental monitoring. CRISPR/Cas systems have emerged as transformative tools in biosensing due to their unparalleled specificity, programmability, and unique enzymatic activities. They exhibit two key cleavage behaviors: precise ON-target cleavage guided by specific protospacers, which ensures accurate target recognition, and bystander cleavage activity triggered upon target binding, which enables robust signal amplification. These properties make CRISPR/Cas systems highly versatile for designing biosensors for ultra-sensitive detection. This review comprehensively explores recent advancements in CRISPR/Cas system-based biosensors, highlighting their impact on improving biosensing performance. We discuss the integration of CRISPR/Cas systems with diverse signal readout mechanisms, including electrochemical, fluorescent, colorimetric, surface-enhanced Raman scattering (SERS), and so on. Additionally, we examine the development of integrated biosensing systems, such as microfluidic devices and portable biosensors, which leverage CRISPR/Cas technology for point-of-care testing (POCT) and high-throughput analysis. Furthermore, we identify unresolved challenges, aiming to inspire innovative solutions and accelerate the translation of these technologies into practical applications for diagnostics, food, and environment safety.

Thymol inhibits ergosterol biosynthesis in Nakaseomyces glabratus, but differently from azole antifungals.

Thymol inhibits ergosterol biosynthesis in Nakaseomyces glabratus, but differently from azole antifungals.

Role of Artificial Intelligence in Drug Discovery to Revolutionize the Pharmaceutical Industry: Resources, Methods and Applications.

Traditional drug discovery methods such as wet-lab testing, validations, and synthetic techniques are time-consuming and expensive. Artificial Intelligence (AI) approaches have progressed to the point where they can have a significant impact on the drug discovery process. Using massive volumes of open data, artificial intelligence methods are revolutionizing the pharmaceutical industry. In the last few decades, many AI-based models have been developed and implemented in many areas of the drug development process. These models have been used as a supplement to conventional research to uncover superior pharmaceuticals expeditiously. AI's involvement in the pharmaceutical industry was used mostly for reverse engineering of existing patents and the invention of new synthesis pathways. Drug research and development to repurposing and productivity benefits in the pharmaceutical business through clinical trials. AI is studied in this article for its numerous potential uses. We have discussed how AI can be put to use in the pharmaceutical sector, specifically for predicting a drug's toxicity, bioactivity, and physicochemical characteristics, among other things. In this review article, we have discussed its application to a variety of problems, including de novo drug discovery, target structure prediction, interaction prediction, and binding affinity prediction. AI for predicting drug interactions and nanomedicines were also considered.

De novo discovery of cyclic peptide inhibitors of IL-11 signaling

De novo discovery of cyclic peptide inhibitors of IL-11 signaling

DeepAptamer: Advancing high-affinity aptamer discovery with a hybrid deep learning model.

DeepAptamer: Advancing high-affinity aptamer discovery with a hybrid deep learning model.

Integration of single-cell RNA sequencing and network pharmacology to elucidate the effect of Yantiao Formula on alleviating ALI by regulating the polarization of alveolar macrophages.

Integration of single-cell RNA sequencing and network pharmacology to elucidate the effect of Yantiao Formula on alleviating ALI by regulating the polarization of alveolar macrophages.

Chikusetsu saponin IVa attenuates aging by improving autophagy and mitophagy.

Chikusetsu saponin IVa attenuates aging by improving autophagy and mitophagy.

Exploring Fluorescence Spectral Shifts in Aptamer-Intercalating Cyanine Dye Complexes upon Binding to Specific Small Molecules.

DNA intercalating cyanine dyes, such as SYBR Green I (SG) and OliGreen (OG), are widely used in developing label-free, fluorescent aptamer-based biosensors. Despite their widespread use for direct analyte detection through changes in fluorescence intensity, the effects of dye concentrations and the specific nature of their interactions have been inadequately explored. Here, we reported how dye-to-base ratios (dbrs) influence the fluorescent response of DNA intercalating dyes in aptamer systems targeting adenosine triphosphate (ATP) and l-argininamide (LAA). We initially examined the fluorescence spectral shifts of an ATP aptamer (ABA) with SG across varying dbrs, observing an emission shift to longer wavelengths as the dbrs increased. Subsequently, systematic analysis of the ATP aptamer and SG complex (ABA/SG) at different target concentrations revealed a "signal-off" phenomenon at a very low dbr of 0.1, which transitioned to a blue shift in the fluorescence spectra at higher dbr values of 0.7 and 2.0. Further extending our research, we explored the use of OG as a ratiometric probe for detecting l-argininamide, noting similar spectral shifts to shorter wavelengths upon target binding. Absorption spectroscopy, circular dichroism (CD), and meticulously designed control studies were employed to elucidate the spectral shift phenomenon comprehensively. Our findings underscore the significant impact of dye selection and concentration on the performance of fluorescence aptasensors and demonstrate that clear spectral shifts, indicative of target binding, occur upon binding to targets, particularly at higher dye loading; however, excessive dye concentrations can perturb the aptamer structure, reducing its binding affinity. We believe that our findings will provide new insights into designing aptamer-based fluorescence assays for the sensitive and specific detection of small molecules.

Protective effect of Pinacidil on hypoxic-reoxygenated cardiomyocytes in vitro and in vivo via HIF-1α/HRE pathway.

Cardiomyocyte hypoxia-reoxygenation (HR) is considered as a major cause of heart failure. Pinacidil is a classic ATP sensitive potassium channel opener and plays a crucial role in cardiomyocyte HR injuries. However, the specific mechanism is poorly understood. We established HR rat model and introduced 5-Hydroxydecanoate (5-HD), N-(2-Mercaptopropionyl)-glycine (MPG), and Dimethylethylenediylglycine (DMOG) to investigate the protection of Pinacidil (P) oncardiomyocyte. HE staining, electron microscopy and JC-1 staining were used to observe mitochondrial structure and mitochondrial membrane potential (MMP). Reactive oxygen species (ROS), hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor A (VEGF-A), heme oxygenase-1 (HO-1), and induced nitric oxide synthase (iNOS) were analyzed in this study. Network pharmacology analysis and auto-docking were used to predict the possible target of Pinadicil under cardiomyocyte HR condition. The integrity of mitochondrial structure and MMP were effectively promoted in P and MPG+DMOG + P groups. ROS was significantly increased after HR, treatment with P or MPG+DMOG + P, the content of ROS was increased. The expressions of HIF-1α, VEGF-A, HO-1 and iNOS were significantly increased in P and MPG+DMOG + P groups compared with HR group. Docking results confirmed that prolyl hydroxylase (PHD) was the most possible target for unsaturated binding with Pinacidil guanidine. Altogether, these data indicate that Pinacidil up-regulated and activated HIF-1α protein to protect caridomyocytes against HR injuries and the mechanism may be related to Pinacidil guanidine binding to PHD.

Prostate Cancer: De-regulated Circular RNAs With Efficacy in Preclinical In Vivo Models.

Therapy resistance, including castration-resistance and metastasis, remains a major hurdle in the treatment of prostate cancer. In order to identify novel therapeutic targets and treatment modalities for prostate cancer, we conducted a comprehensive literature search on PubMed to identify de-regulated circular RNAs that influence treatment efficacy in preclinical prostate cancer-related in vivo models. Our analysis identified 49 circular RNAs associated with various processes, including treatment resistance, transmembrane and secreted proteins, transcription factors, signaling cascades, human antigen R, nuclear receptor binding, ubiquitination, metabolism, epigenetics and other target categories. The identified targets and circular RNAs can be further scrutinized through target validation approaches. Down-regulated circular RNAs are candidates for reconstitution therapy, while up-regulated RNAs can be inhibited using small interfering RNA (siRNA), antisense oligonucleotides (ASO) or clustered regularly interspaced short palindromic repeats/CRISPR associated (CRISPR-CAS)-related approaches.

Using fluorescently labeled wheat germ agglutinin to track lipopolysaccharide transport to the outer membrane in Escherichia coli.

The cell envelope of gram-negative bacteria consists of two membranes sandwiching the peptidoglycan (PG) cell wall. The outer membrane (OM) contains integrated beta-barrel proteins and has an outer leaflet composed of lipopolysaccharide (LPS). LPS is transported from the inner membrane where it is made to the OM surface by the Lpt system. In the polarly elongating alpha-proteobacterium Brucella abortus, LPS transport has been localized to the polar growth zone and division site. However, LPS transport has not been tracked in live proteobacteria like Escherichia coli that elongate by dispersed incorporation of envelope material along their cell body. Here, we report an investigation into the binding target of fluorescently labeled wheat germ agglutinin (FL-WGA) on E. coli cells that led to the development of a method for visualizing LPS transport. We show that instead of PG or enterobacterial common antigen for which FL-WGA labeling has been used to detect in the past, this probe recognizes LPS modified with a terminal N-acetylglucosamine formed by the defective O-antigen synthesis pathway of laboratory strains of E. coli. This finding enabled the construction of mutants inducible for LPS modification that were used together with FL-WGA labeling to track LPS transport to the cell surface. We show that new LPS is inserted throughout the cell cylinder and at the division site, but not at the cell poles. A similar pattern was observed previously for PG synthesis and OM protein insertion in E. coli, suggesting that LPS transport to the OM is coordinated with these processes.IMPORTANCEGram-negative bacteria like Escherichia coli are surrounded by a multilayered cell envelope that includes an outer membrane (OM) responsible for their high intrinsic resistance to antibiotics. The outer leaflet of this membrane is composed of a glycolipid called lipopolysaccharide (LPS). Here, we report the development of an imaging method to track the transport of LPS to the E. coli outer membrane. The results indicate that transport occurs throughout the cell cylinder and at the division site, but not at the cell poles. A similar pattern was observed previously when cell wall synthesis and the insertion of proteins into the OM were tracked. Our results therefore suggest that LPS transport to the OM is coordinated with other essential processes that underly gram-negative cell envelope biogenesis.

CTCF regulates global chromatin accessibility and transcription during rod photoreceptor development

Chromatin architecture facilitates accurate transcription at a number of loci, but it remains unclear how much chromatin architecture is involved in global transcriptional regulation. Previous work has shown that rapid depletion of the architectural protein CTCF in cell culture alters global chromatin organization but results in surprisingly limited gene expression changes. This discrepancy has also been observed when other architectural proteins are depleted, and one possible explanation is that full transcriptional changes are masked by cellular heterogeneity. We tested this idea by performing multiomics analyses with sorted juvenile postmitotic mouse rods, which undergo synchronized development, and we identified CTCF-dependent regulation of global chromatin accessibility and gene expression. CTCF depletion leads to dysregulation of ~20% of the entire transcriptome (>3,000 genes) and ~41% of genome accessibility (>27,000 sites) before any prominent cellular or physiological phenotypes arise. Importantly, these changes are highly enriched for CTCF occupancy at euchromatin, suggesting direct CTCF binding and transcriptional regulation at these active loci. CTCF mainly promotes chromatin accessibility and frequently inhibits expression of these direct binding targets, which are enriched for binding motifs of transcription repressors. These findings provide different and sometimes opposite conclusions from previous studies, emphasizing the need to consider cellular heterogeneity and cell-type specificity when performing multiomics analyses. CTCF knockout rods undergo complete degeneration by adulthood, indicating an essential role for their viability. We conclude that the architectural protein CTCF binds chromatin and regulates global chromatin accessibility and transcription during rod development.

Intrinsically disordered regions as facilitators of the transcription factor target search.

Transcription factors (TFs) contribute to organismal development and function by regulating gene expression. Despite decades of research, the factors determining the specificity and speed at which eukaryotic TFs detect their target binding sites remain poorly understood. Recent studies have pointed to intrinsically disordered regions (IDRs) within TFs as key regulators of the process by which TFs find their target sites on DNA (the TF target search). However, IDRs are challenging to study because they can confer specificity despite low sequence complexity and can be functionally conserved despite rapid sequence divergence. Nevertheless, emerging computational and experimental approaches are beginning to elucidate the sequence-function relationship within the IDRs of TFs. Additional insights are informing potential mechanisms underlying the IDR-directed search for the DNA targets of TFs, including incorporation into biomolecular condensates, facilitating TF co-localization, and the hypothesis that IDRs recognize and directly interact with specific genomic regions.

In vitro Stability Study of a Panel of Commercial Antibodies at Physiological pH and Temperature as a Guide to Screen Biologic Candidate Molecules for the Potential Risk of In vivo Asparagine Deamidation and Activity Loss.

Biologic drug molecules such as antibodies are exposed to the physiological stress conditions of pH 7.4 and 37°C during their long circulation lifetime in vivo. The stress on biologic molecules in vivo is more severe compared to that under typical storage conditions of low pH formulation and cold temperature. Chemical degradation of critical residues such as asparagine may occur in vivo, leading to potential loss of biological activity. This study describes a physiologically relevant and convenient in vitro PBS stress condition of pH 7.4 and 40°C for pre-clinical stability screening of biologic molecules. As benchmarks, multiple commercial antibodies (alirocumab, evolocumab, golimumab, ramucirumab, and trastuzumab) were tested in parallel for formulation stability at storage and accelerated temperature conditions and for physiological stability at pH 7.4 and 40°C stress both for 3-4weeks. The stressed antibodies were monitored for chemical modification and target binding, without requiring affinity purification. The major CDR chemical modifications observed in PBS-stressed commercial antibodies were deamidations of asparagine residues. Although slight decreases in target binding were observed for two antibodies, the affinities overall remained strong after PBS stress. This benchmarking study of commercial antibodies would be useful as a guide to screen discovery-stage biologic molecules both for drug product stability at formulation pH under storage and accelerated temperature conditions and for physiological stability under in vivo-mimicking pH and temperature stress condition.

IL13Rα2-Targeting Antibodies for Immuno-PET in Solid Malignancies.

Interleukin-13 receptor α-2 (IL13Rα2) is a cell surface receptor frequently expressed in solid malignancies, such as glioblastoma and melanoma, with limited expression in healthy tissue, rendering it an ideal target for noninvasive and specific tumor delineation. In this study, we report the development of 5 novel IL13Rα2-targeted human monoclonal antibodies (mAbs) KLG-1-5; in subsequent invitro and invivo studies after radiolabeling with 89Zr, we evaluate their performance to identify a lead candidate. Methods: Five novel human anti-IL13Rα2 mAbs KLG-1-5 were developed and invitro binding properties and target specificity assessed. In vivo 89Zr-immuno-PET using KLG-1-5 was conducted in a subcutaneous U-87 MG glioblastoma mouse model, and a mass dose titration study was conducted with lead candidate KLG-3. Ex vivo biodistribution results were used to derive prospective dosimetry of 177Lu-labeled KLG-3. Targeting with KLG-3 was also verified in an A-375 melanoma model using the optimized conditions determined in the U-87 MG xenograft model. Results: In vitro studies confirmed target specificity and pico- to low nanomolar binding affinity. Immuno-PET studies with KLG-1-5 in U-87 MG xenografts demonstrated continuously increasing tumoral uptake with maximal uptake at 144 h after tracer injection, clearance of the unbound tracer from the blood pool, and little uptake in any other normal tissues, leading to high-contrast images. KLG-3 provided the highest tumoral uptake and tumor-to-normal tissue ratios and was chosen as the lead candidate, and further dose optimization with this antibody led to tumoral uptake of 97 ± 6 maximum percent of injected dose per gram at 144 h after tracer injection. Ex vivo biodistribution-derived prospective dosimetry for 177Lu-labeled KLG-3 predicted a favorable therapeutic index, encouraging the development of IL13Rα2-targeted radioimmunotherapy. Of note, KLG-3 performed similarly well in a melanoma model, emphasizing the versatility of this antibody. Conclusion: Lead candidate anti-IL13Rα2 mAb KLG-3 validated highly specific target binding in human glioblastoma and melanoma models, resulting in high-contrast PET images with minimal accumulation in off-target healthy tissues. Prospective dosimetry of its 177Lu-labeled counterpart suggested therapeutic efficacy at relatively low injected activities, supporting further pursuit of KLG-3 in future translational radioimmunotherapy applications.

Core-Extended Naphthalene Diimide Dyads as Light-Up Probes with Targeted Cytotoxicity Toward Tumor Cells.

Within the framework of rational drug design, this study introduces a novel approach to enhance the specificity of small molecules in targeting cancer cells. This approach starts from the use of dyads merging into a single entity, a naphthalene diimide (NDI) and core-extended NDI (ceNDI), both known as G-quadruplex (G4) ligands and fluorescent probes. The strategy aims to leverage the unique diagnostic strengths of the ceNDI moiety featuring red emission by improving its binding affinity and target selectivity through inclusion in dyads built with different linkers. The newly developed NDI-ceNDI dyads are promising probes, as they exhibit fluorescence turn-on upon DNA recognition and induced circular dichroism signals dependent on DNA conformation. Both dyads have an excellent affinity for hybrid G4, with two orders of magnitude higher binding constants than those for ds DNA. Their high cytotoxicity on cancer cell lines further demonstrates their potential as therapeutic agents, highlighting the role of the linker in target selectivity. Specifically, only the dyad with the rigid triazole linker exhibits selectively induced DNA damage in transformed cells, compared to normal cells primarily targeting telomeric regions. Our findings shed light on DIPAC's potential as a promising theranostic agent, offering insights into future developments in precision medicine.

S100A4 participates in sepsis-induced endothelial cell inflammatory response and barrier damage by binding to BRD4

Background Research has shown that S100A4 is upregulated in endothelial cells when exposed to serum from septic patients. This article aims to explore the role of endogenous S100A4 in lipopolysaccharide (LPS)-induced endothelial cells. Methods A septic HUVECs injury model was established using LPS and transfected with siRNA-S100A4 or Ov-BRD4 plasmid. Targets of S100A4 were predicted using online databases, and immunoprecipitation (IP) was used to verify the binding of S100A4 and targets. Cell viability, levels of apoptosis, and the expression of apoptosis-related proteins were measured to assess cell injury. Transendothelial electrical resistance (TER) and the expression of tight junction proteins were measured to assess cell barrier function. Assessed the inflammatory response by measuring the levels of inflammatory factors, the adhesion of THP-1 monocytes, and the expression of adhesion molecules. Results Database prediction and IP verification indicated that S100A4 could bind to BRD4 in LPS-induced HUVECs, and the expression of S100A4 and BRD4 was increased in LPS-induced HUVECs. Interference with S100A4 significantly enhanced the cell viability and TER, reduced the apoptosis, TNFα, IL-1β, and IL-6 levels, and THP-1 adhesion number in LPS-treated HUVECs. Additionally, interference with S100A4 upregulated the expression of Bcl2, ZO-1, occludin, and claudin-4 proteins, and downregulated the expression of BRD4, Bax, cleaved caspase-3, ICAM-1, VCAM-1, and E-selectin proteins in LPS-induced HUVECs. However, overexpression of BRD4 significantly attenuated the protective effect of interfering with S100A4 on LPS-induced HUVECs. Conclusion S100A4 is involved in LPS-induced inflammatory response and barrier damage in HUVECs by binding to BRD4.