G protein-coupled receptors (GPCRs) are a big family of membrane proteins which represent one of the main classes of drug targets. However, their investigation presents several challenges, among which their instability outside the membrane environment. Different strategies for the drug discovery of this target are available, and surface plasmon resonance (SPR) stands out as one of the most informative and widespread binding assays, with many advantages such as real-time and label-free analyses resulting in the definition of both affinity and kinetic constants. This review covers the applications of SPR in GPCR drug discovery of the last 10 years and classifies the papers based on the immobilization strategy on the SPR sensor chip to maintain receptor stability. In particular, GPCR immobilization can occur in its native membrane by immobilizing whole cells or membrane fragments, using membrane mimetics (such as lipoparticles, lentiviral particles, liposomes, lipoproteins, nanodiscs, or planar lipid membranes) or immobilizing the isolated receptor stabilized by the use of detergents or engineering approaches. Different examples were considered and pros and cons of each strategy were presented.

The characterization of drug-target interactions is a key component of drug discovery, testing, and development. Affinity chromatography is one approach that can be used for this type of analysis. For instance, this may be done by using an immobilized target as a stationary phase and a drug as the applied solute. This review will discuss the various ways in which affinity chromatographic methods have been used to examine drug-target interactions, with an emphasis on high-performance methods. The general principles of this approach and factors to consider in its use for drug-target interaction analysis will first be examined. Methods based on zonal elution or frontal analysis for binding and competition studies will then be discussed. Various techniques for kinetic studies will next be considered, along with approaches that employ secondary binding agents and hybrid techniques. In each case, the general principles and theory of an approach will be given along with examples of its use in drug-target interaction studies. Advantages or limitations of each approach will be provided as well. This information should make it possible in the future to extend these techniques to other drug-target systems of interest in biomedical research and drug testing or development.

Affinity selection mass spectrometry (AS-MS) has emerged as a powerful label-free technique for identifying and characterizing ligand-target interactions. This review explores the diverse applications of AS-MS in drug discovery, including its role in selective screening, binding site characterization, and quantitative affinity determination. We discuss the use of AS-MS for determining equilibrium dissociation constants (K_D) and competitive binding parameters (affinity competition experiment 50% (ACE₅₀)), highlighting its ability to rank ligand affinities efficiently. The review also examines AS-MS applications in fragment-based drug discovery (FBDD), screening for molecular glues, and investigating interactions with membrane proteins. Moreover, we address key technical challenges, including competitive binding effects, protein stability, and ligand dissociation kinetics, along with recent advancements in automation and artificial intelligence (AI) integration. Rather than providing a comprehensive literature review, this work aims to broaden the applicability of AS-MS assays and encourage researchers to explore its use in underutilized contexts. By providing rapid and high-sensitivity affinity measurements, AS-MS continues to expand its role in drug discovery and structural biology, complementing conventional biophysical techniques.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening technology is redefining the landscape of drug discovery and therapeutic target identification by providing a precise and scalable platform for functional genomics. The development of extensive single-guide RNA (sgRNA) libraries enables high-throughput screening (HTS) that systematically investigates gene-drug interactions across the genome. This powerful approach has found broad applications in identifying drug targets for various diseases, including cancer, infectious diseases, metabolic disorders, and neurodegenerative conditions, playing a crucial role in elucidating drug mechanisms and facilitating drug screening. Despite challenges like off-target effects, data complexity, and ethical or regulatory concerns, ongoing advancements in CRISPR technology and bioinformatics are steadily overcoming these limitations. Additionally, by integrating with organoid models, artificial intelligence (AI), and big data technologies, CRISPR screening expands the scale, intelligence, and automation of drug discovery. This integration boosts data analysis efficiency and offers robust support for uncovering new therapeutic targets and mechanisms. This review outlines the fundamental principles and applications of CRISPR screening technology, delves into specific case studies and technical challenges, and highlights its expanding role in drug discovery and target identification. It also examines the potential for clinical translation and addresses the associated ethical and regulatory considerations.

Aptamer therapeutics represent a class of target-based therapies that leverage their high specificity and affinity for diverse molecular targets. As single-stranded DNA or RNA oligonucleotides, aptamers offer advantages in therapeutic applications. A critical aspect of aptamer drug development is the selection process, which has seen significant advancements through various in vitro selection methods, including Systematic Evolution of Ligands by Exponential Enrichment and its emerging variations. Recent progress has also introduced functional screening strategies that directly identify pharmacologically active aptamers, accelerating drug discovery. The applications of aptamers in disease treatment are expanding across oncology, neurodegenerative disorders, infectious diseases and other diseases. Aptamers exhibit versatile mechanisms of action, including blocking interactions, recruiting protein machinery, and inhibiting target functions. By addressing key limitations and presenting future directions, this review provides a comprehensive perspective on the recent evolving landscape of aptamer technology and its transformative potential in modern medicine.

Advancements in tumor immunotherapy highlight the significant potential of antibody drugs, a key category of biological agents, for treating cancer and autoimmune diseases. This paper begins by defining and classifying key targets in tumor immunity, as well as discussing their structural and functional characteristics. Subsequently, it elaborates on innovative technologies for antibody drug screening, which, when integrated with contemporary molecular biology, biotechnology, and computational biology, have substantially enhanced the efficiency and accuracy of target identification and antibody drug screening processes. Despite the promising prospects of tumor immunotherapy, certain limitations persist in its practical implementation. In conclusion, this paper offers a comprehensive examination of the cutting-edge developments in tumor immunotherapy, focusing on the aspects of tumor immunotherapy itself, critical targets for immunotherapy, and novel technologies and methodologies for antibody screening. This analysis is crucial for advancing the field of tumor immunotherapy and for enhancing both therapeutic efficacy and safety. Furthermore, research and development (R&D) of antibody drugs in other domains, such as autoimmune and inflammatory diseases, can benefit from it.

G protein-coupled receptors (GPCRs), the largest superfamily of cell surface receptors and targets for over 30% of current clinical drugs, remain crucial for future therapeutic development. This study introduces a novel NanoLuciferase (NanoLuc, Nluc) bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay, utilizing the gonadotrophin-releasing hormone (GnRH) receptor as a model system. Our study demonstrates that sulfo-cyanine 5 (sCy⁵) is an ideal fluorophore compatible with NanoBRET, enabling sensitive measurement of ligand binding on living cell membranes. A novel GnRH analogue, sCy⁵-D-Lys⁶-GnRH, was synthesized by conjugating sCy⁵ on the substituted D-Lys⁶ of the native GnRH I. Substitution of Gly⁶ of GnRH I with sCy⁵-D-Lys⁶ stabilizes the βII’ turn configuration of the decapeptide that exhibits high affinity and specificity for GnRH receptors while maintaining agonist activity. To address the characteristically low expression of the human GnRH receptor (hGnRHR), we engineered a modified receptor by fusing NanoLuc with an interleukin-6 (IL6) secretory signal peptide (secNluc) to the N-terminus of the hGnRHR and deleting Lys191 (K191Δ) within the 2nd extracellular loop. This modification, N-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor with K191 deletion (N-secNluc-hGnRHR-K191Δ) significantly enhances receptor expression without altering ligand binding affinity, resulting in a robust BRET signal detection (Z' ≥ 0.5) between sCy⁵-D-Lys⁶-GnRH and the modified receptor. Our innovative approach using sCy⁵ to conjugate ligands offers several key advantages: high sensitivity and specificity, remarkably low non-specific binding (NSB), compatibility with live-cell assays, and suitability for high-throughput drug screening, which may accelerate the discovery of new therapeutics for GnRH receptor signal-selective drugs and potentially for other GPCRs.

The natural killer (NK) group 2 member A/C-type lectin domain family 4 member A (NKG2A/CD94) heterodimeric receptor is commonly recognized as a crucial immune checkpoint in NK cells. Currently, there is a notable lack of small-molecule inhibitors specifically targeting NKG2A that have progressed to clinical trials, and established screening methodologies for identifying such inhibitors remain limited. Cell membrane chromatography (CMC) is a biochromatographic technique that leverages the specific interactions between membrane receptors and their ligands. In this study, a comprehensive two-dimensional (2D) NKG2A/CD94 and HEK293 CMC comparative analysis system was developed to screen for selective NKG2A/CD94 ligands derived from Echinacea purpurea (L.) Moench (EP) and Alpinia katsumadai Hayata (AKH). The comprehensive 2D CMC comparative analysis system demonstrated superior selection performance, resulting in the successful screening and identification of five compounds. Of these compounds, chicoric acid and alpinetin exhibited greater binding affinity for the NKG2A/CD94 CMC column compared to the HEK293 CMC column, leading to their selection for further efficacy verification. Surface plasmon resonance (SPR) analysis revealed that chicoric acid and alpinetin exhibit binding affinities of 12.9 and 9.49 μM, to NKG2A/CD94. Molecular docking analyses and pharmacological investigations further demonstrated that both compounds could influence NK cell activation by interacting with the NKG2A/CD94. These findings suggest their potential as novel NKG2A/CD94 immune checkpoint inhibitors (ICIs). Additionally, the comprehensive 2D CMC system serves as a robust and practical platform for drug discovery, and could be applied to other immune checkpoint receptor models.

Cell membrane coating technology has recently emerged as a promising platform for drug activity assessment due to its unique biointerfacing capabilities. Nevertheless, its integration with conventional detection methods such as high performance liquid chromatography (HPLC) and fluorescence probe analysis remains limited by poor specificity and low accuracy, primarily resulting from non-specific adsorption of non-target membrane receptors and interference from background signals. In this study, we presented a collaborative strategy that integrates aptamers with cell membrane coating technology to establish a novel electrochemiluminescence (ECL)-DNA biosensor platform for specifically detecting drug-target receptor interactions. High specificity was achieved through competitive binding between aptamers and drug candidates for membrane receptors, while high accuracy was ensured by employing an ECL detection system incorporating signal cascade amplification and three-dimensional (3D) DNA walkers, enabling reliable performance even with complex biological samples. Using this approach, we demonstrated a linear dynamic range of 1 nmol/L to 2 μmol/L for the detection of desloratadine activity, with a limit of detection (LOD) of 0.16 nmol/L. Furthermore, the platform was successfully applied to evaluate the binding activity of eight drugs to angiotensin-converting enzyme 2 (ACE2), and their pharmacological activities were further characterized. Overall, this aptamer-cell membrane coating synergistic strategy offered excellent specificity and ultra-high sensitivity, making it a valuable tool for elucidating drug-receptor mechanisms of action and providing a robust reference for preclinical drug activity evaluation.

Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disorder with an increasing incidence worldwide. Due to the complex and unclear therapeutic targets, unmet UC therapeutic drugs still exist. Recently, acylcarnitine metabolism disorder has been linked to intestinal inflammation, but its role in UC remains elusive. According to our preliminary non-targeted metabolomics data, acylcarnitines (ACs) was screened as the disturbed metabolites in the different intestinal inflammation-related diseases. Here we quantified 26 ACs within liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the dextran sulfate sodium (DSS)-induced UC rat model, and found that long-chain acylcarnitines (LCACs) were increased to varying degrees. As the key metabolites of fatty acid β-oxidation (FAO), the upstream metabolites long-chain fatty acids (LCFAs) and the related metabolic enzymes were further characterized, the results showed that the rate-limiting enzyme carnitine palmitoyltransferase 1A (CPT1A)-mediated LCFAs-LCACs metabolic axis was activated sharply. Next in vitro experiments exhibited that CPT1A was significantly upregulated in both inflammatory macrophages and colonic epithelial cells, and inhibition or knockdown of CPT1A could reduce the inflammation level remarkably. Thus, we screened the pharmacologic inhibitors of CPT1A from US Food and Drug Administration (FDA) approved drugs, within molecular docking, Western blot and cell membrane chromatography (CMC) technology, gliquidone was found to inhibit CPT1A in a dose-dependent manner and exert anti-inflammatory effects in vitro. Animal experiments also showed that gliquidone alleviated DSS-induced UC significantly. In summary, our study presents that within metabolomics analysis, inhibiting CPT1A is focused to be a potential therapeutic strategy against UC, and gliquidone represents an alternative treatment.

The N-terminal domain of influenza viral polymerase (PA_N), a highly conserved region with critical catalytic function related to viral RNA replication and transcription, is considered as a very promising anti-influenza drug target. There is an urgent need for highly efficient and rapid screening methods to identify potential PA_N inhibitors (PA_NIs) from complex matrices. In this work, a novel high-throughput screening (HTS) platform was established by coupling high performance liquid chromatography and high-resolution mass spectrometry (HPLC-HRMS) with a fluorescence resonance energy transfer (FRET)-based endonuclease activity assay through an at-line nanofractionation (ANF) system. The proposed screening platform could rapidly identify potential PA_NIs from plant extracts with good sensitivity (baloxavir at the half maximal inhibitory concentration (IC₅₀) could be detected) and reliability (Z’ factor of 0.77). This platform was then successfully applied to the screening of potential inhibitors against PA_N/PA_N I38T from an aqueous extract of Artemisiae Argyi Folium and 17 potential PA_NIs were identified. Among them, three compounds (cynarine, isochlorogenic acid B, and isochlorogenic acid C) showed comparable inhibitory activity against PA_N and even better activity against PA_N I38T, compared to baloxavir. This study not only established a novel high-throughput ANF-based PA_NIs screening platform, but also proved the feasibility to discover PA_NIs from complex traditional Chinese medicines (TCMs), which has a great potential in future anti-influenza drug discovery.

The main protease (M^pro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been validated as a therapeutic target for antiviral drug development, given its critical role in the viral life cycle. SARS-CoV-2 M^pro contains 12 cysteine residues, which are susceptible to covalent modification by nucleophilic entities. In this study, we showcase an efficient strategy to uncover the key covalent inhibitors of SARS-CoV-2 M^pro from herbal extracts and decipher their synergistic anti-M^pro mechanisms. Preliminary screening identified Rhodiola crenulata root (RCR), a well-known Tibetan herb, showing the most potent time-dependent inhibition against SARS-CoV-2 M^pro. By integrating fluorescence resonance energy transfer (FRET)-based biochemical assay with phytochemical and chemoproteomic profiling, we efficiently identified thirteen M^pro covalent inhibitors from the crude extract of RCR. Among these, rhodiosin and gallic acid were validated as the key anti-M^pro constituents, due to their strong anti-M^pro effects and high abundance in RCR. Remarkably, their combination exhibited a pronounced synergy in M^pro inhibition. Further intact protein mass measurements and top-down mass spectrometry (MS) analysis, complemented by biophysical methods, elucidated how these two compounds work in concert. Our findings revealed that rhodiosin functions as an allosteric inhibitor, disrupting M^pro dimerization and significantly facilitating the covalent modification of M^pro by gallic acid. Collectively, the covalent SARS-CoV-2 M^pro inhibitors are efficiently identified from a Tibetan herb, while a phytochemical combination with synergistic anti-M^pro effects and their unique allosteric-induced cooperative modification mechanism are revealed.

Src homology 2 domain-containing phosphatase 2 (SHP2) is a pivotal regulator linking receptor tyrosine kinase (RTK) signaling. Abnormal SHP2 activity has been associated with tumorigenesis and metastasis. Although some SHP2-targeting modulators have entered clinical trials, U.S. Food and Drug Administraction (FDA)-approved SHP2 targeting drugs are still not available. Herein, we describe cooperative biochemical inhibition experiments that facilitate the identification of both catalytic and allosteric SHP2 inhibitors using an in-house natural product (NP) library. Based on this screening methodology, structurally diverse sets of NPs were characterized, among which dihydrotanshinone I (DHT) potently inhibited the wild-type SHP2 protein tyrosine phosphatase (PTP) domain and gain-of-function SHP2 variants. Trichostatin A (TSA) bound to the “tunnel” binding site, acting as an allosteric inhibitor. This study illustrates an optimized screening methodology and tactics to identify novel SHP2 modulators from NPs and provides a foundation for further NP-based drug development for the treatment of RTK-driven cancer.

Osteoporosis, a severe systemic skeletal disorder characterized by decreased bone mineral density, leads to increased risks of bone fragility and fracture. Although some herbal medicines (HMs) are clinically used for treating osteoporosis, the crucial anti-osteoporotic constituents and their mechanisms have not been well-elucidated. Notum, a negative regulator of Wnt/β-catenin signaling, has been validated as a druggable target for enhancing cortical bone thickness and alleviating osteoporosis. Herein, we showcase an efficient strategy for uncovering the key anti-Notum constituents from HMs via integrating biochemical, phytochemical, computational, and cellular assays. Following screening the anti-Notum potentials of HMs, Polygonum multiflorum Thunb. (PM), a commonly used anti-osteoporosis herb, showed potent and competitive inhibition against Notum. Phytochemical profiling coupling with docking-based virtual screening suggested that three anthraquinones, including rhein, emodin, and chrysophanol, showed high binding-potency towards Notum. Biochemical assays validated that three anthraquinones were strong competitive inhibitors of Notum, while rhein was the most potent one (IC₅₀ = 9.98 nM). Cellular investigations demonstrated that rhein markedly promoted osteoblast differentiation in dexamethasone-challenged MC3T3-E1 osteoblasts, while RNA sequencing showed that rhein remarkably regulated Wnt signaling-related and osteogenic differentiation-related genes. In vivo tests showed that rhein displayed favorable safety profiles in healthy mice and this agent significantly elevated bone mineral density, and augmented trabecula and cortical bone thickness in dexamethasone-induced osteoporotic mice. Collectively, this study showcases an efficient strategy for uncovering the key anti-Notum constituents from HMs, while rhein was identified as a naturally occurring Notum inhibitor that shows favorable safety profiles and impressive anti-osteoporosis effects.

Elapid snakebites cause severe toxicity, predominantly neurotoxicity and general cytotoxicity. However, the specific cellular impacts of individual venom toxins remain largely underexplored. This study developed a high-throughput platform for profiling cytotoxicity from elapid venoms, focusing on nanofractionation analytics to enhance selectivity and toxin identification. Elapid Venoms were tested on four human cell lines, representing kidney (RPTEC/TERT1), liver (HepaRG), endothelial (iPSC-EC), and skin (HaCaT) tissues. Cytotoxic effects were assessed through cell coverage, viability, and metabolic assays in both crude and nanofractionated venom samples. Nanofractionation revealed selective cytotoxicity in venom components, notably phospholipases A₂ (PLA₂s) and three-finger toxins (3FTxs), which impaired membrane integrity and cellular metabolism. Crude B. multicinctus venom displayed specific cytotoxicity toward liver and skin cells but not kidney or endothelial cells. Cytotoxicity of nanofractionated B. multicinctus venom was lost, likely due to denaturing conditions of the reversed-phase separation. Fractionation after size exclusion chromatography (SEC) for post-column bioassaying to avoid toxin denaturation yielded bioactive fractions, with 3FTxs, PLA₂s, and Kunitz-type serine protease (KUNs) likely responsible for the observed cell permeability disruption, extracellular matrix (ECM) degradation, and metabolic loss. This integrated analytical workflow, combining nanofractionation with high-throughput cytotoxicity assays and venomics, enabled rapid identification of venom components with cell type-specific toxicity. Our findings contribute to understanding elapid venom toxicity and can aid in developing targeted snakebite treatments focusing on cytotoxicity responsible for tissue-specific damage.

Lung cancer takes the lead in terms of global cancer incidence and mortality rates. 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) serves as a universally conserved energy sensor throughout evolution checkpoint that orchestrates energy balance and metabolic homeostasis. However, AMPK activation has a complex, dual function in both the onset and advancement of lung cancer. Despite its protumorigenic effects, targeting AMPK with inhibitors to suppress cancer progression remains a critical area of research. An innovative high-content screening platform integrating small-molecule microarrays (SMMs) with oblique-incidence reflectivity difference (OI-RD) optical detection was established for AMPK inhibitor discovery. Alterations in the interfacial refractive index revealed that Ribavirin, an antiviral drug, has a high affinity for AMPK. Ribavirin binds directly to AMPK, suppressing its activation in mouse and human cells. By inhibiting AMPK phosphorylation, Ribavirin affects the downstream phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1), thereby regulating tumor cell proliferation and apoptosis. These results identify Ribavirin as a new AMPK inhibitor with potential utility in lung cancer therapy.

Conventional ex vivo drug screening platforms struggle to recapitulate native subcellular microenvironments, leading to high off-target rates and compromised discovery of bioactive compounds. To address this, we developed subcellular target-tracking fluorescent-visualization-based interaction screening (SubTrack-FVIS), a platform combining super-resolution imaging with target-specific fluorescent tagging. SubTrack-FVIS first maps nanoscale spatial distributions of drug targets within living cells, then screens compound libraries to identify molecules specifically binding to target-enriched domains, and finally quantifies drug-target interactions through super-resolution imaging tracking. Compared to traditional toolbox, SubTrack-FVIS reduces off-target effects by evaluating compound binding within native subcellular architectures. When applied to the lysosomal vacuolar H⁺-ATPases (V-ATPase) subunit, ATP6V1A, a validated anti-cancer target, this approach identified for lysosomal alkalization fluorescent drug (LAFD) as a potent inhibitor. Super-resolution imaging revealed LAFD's dynamic binding to ATP6V1A clusters, enabling real-time visualization of V-ATPase inhibition and subsequent lysosomal destabilization. Crucially, SubTrack-FVIS uncovered LAFD's unique mechanism of blocking autophagosome-lysosome fusion, resolving autophagic flux obstruction at sub-100 nm resolution. This platform establishes a visualization framework for discovering drugs within physiological subcellular contexts while simultaneously decoding their mechanistic impacts, offering application potential for target-centric drug development.