Multidisciplinary cutting-edge technologies accelerating target-based drug discovery

Drug discovery has begun to move beyond traditional targets, turning instead to more complex target landscapes. These complex targets, including membrane proteins, are characterized by their environment and conformational mobility. A limitation of traditional in vitro screening is that the target protein is not in its native environment. As a result, there is a need to go beyond current methods, this will allow you to study a target proteins functional activity in their native environment. To this end, target-based drug screening technologies have evolved through the convergence of multiple fields including analytical chemistry [1], biophysics [2], cell biology [3], and computational science [4]. This integration can move drug discovery beyond isolated targets and allow for the target protein analysis in their native environment.

In target-based drug screening, affinity-based capture and functional characterization strategies are fundamental to characterize the binding affinity between biological molecules and their target proteins. This allows for the separation, enrichment, and identification of active components, making them particularly suitable for discovering unknown ligands [5]. For example, in affinity chromatography, target proteins are immobilized onto a stationary phase, allowing for the specific extraction of potential ligands from complex matrices (e.g., cell lysates, serum, or compound libraries), followed by identification and quantification of active components [6]. To better study the native conformation and functional environment of membrane proteins, recent advances have prioritized functional screening technologies based on biomimetic interfaces and living cells (cell membrane chromatography (CMC)). CMC immobilizes membrane fragments expressing targets like immune checkpoint NKG2A/CD94 onto the stationary phase, which preserves the target's native structure and lipid environment, facilitating the screening of specific inhibitors [3]. Furthermore, technologies including NanoBRET and surface plasmon resonance further enable the real-time, label-free quantitation of membrane proteins in living cells or biomimetic interfaces, effectively overcoming protein inactivation issues inherent to traditional purification and/or immobilization methods [2,7]. Notably, the electrochemiluminescence DNA (ECL-DNA) biosensor integrates a natural cell membrane expressing angiotensin-converting enzyme 2 (ACE2) with high-sensitivity electrochemiluminescence detection to evaluate drug-binding activity to ACE2 and the antiviral potential within its biomimetic membrane environment [8].

In the analysis of complex matrices, such as natural product extracts, traditional Chinese medicine (TCM) preparations, or biological venoms, activity-guided fractionation combined with phenotypic screening platforms offer unique capabilities. These platforms couple high-efficiency physical separation (e.g., liquid chromatography, and size-exclusion chromatography) with downstream functional activity evaluation (e.g., enzyme activity assays, and cytotoxicity tests) in an online configuration, establishing a cohesive “separation–analysis–detection” workflow. An online nanoscale activity screening platform was developed to rapidly isolate and identify influenza virus protease inhibitors in Artemisia annua [9]. In addition, an integrated platform utilizing size-exclusion chromatography enabled the identification of a specific protein toxin family to cellular damage phenotypes in cobra venom [10].

To address the highly complex protein environment, an integrated strategy combining artificial intelligence with multilevel biological validation can increase efficiency, by integrating computational tools, such as molecular docking and artificial intelligence with experimental results. An example of this, was the discovery of inhibitors for notum esterase or tyrosine phosphatase Src homology-2 containing protein tyrosine phosphatase (SHP2), where computational screening was used initially to focus on candidate molecules, followed by sequential experiments to validate efficacy and mechanism, thereby identifying potent inhibitors from natural product libraries [4,11]. Moreover, label-free and high-throughput detection techniques such as oblique-incidence reflectivity difference facilitate the rapid primary screening of stable soluble enzymes or signaling proteins, enabling the early identification of potential ligands [12]. This was demonstrated in the study of carnitine palmitoyltransferase 1A, where combining metabolomics-based target discovery, in silico binding mode prediction, and functional validation via CMC identified gliclazide as a potential inhibitor [13].

Functional genomics and macromolecule screening technologies have expanded the frontiers of drug discovery. CRISPR-Cas9 functional genomic screening enables the unbiased identification of novel disease phenotype-related targets across the genome-wide, while simultaneously validating the functional necessity of these candidates [14]. Further, screening platforms for antibodies and aptamers, where single B-cell sequencing, phage display, and systematic evolution of ligands by exponential enrichment (SELEX), have been significantly enhanced by artificial intelligence (AI)-assisted design, resulting in the optimization of both the development efficiency and targeting precision of biological drugs [15,16].

Targeted drug screening technologies are evolving toward live-cell and in situ observation, dynamic visualization, with cutting-edge super-resolution imaging technologies. With continued advances in imaging subcellular microenvironments and in vivo systems, targeted drug discovery is transitioning to studying proteins in their physiological environments, which facilitates the in situ analysis of drug localization within specific subcellular structures and possible elucidation of the mechanistic processes [17]. Consequently, these capabilities offer an unprecedented spatiotemporal lens for understanding drug action, while reinforcing that precise site targeting and conformational dynamics are central to future discovery. Ultimately, this evolution propels the field toward greater specificity and the comprehensive characterization of drug-target interactions.