Programmed death-1/programmed death ligand-1 (PD-1/PD-L1) based immunotherapy is a revolutionary cancer therapy with great clinical success. The majority of clinically used PD-1/PD-L1 inhibitors are monoclonal antibodies but their applications are limited due to their poor oral bioavailability and immune-related adverse effects (irAEs). In contrast, several small molecule inhibitors against PD-1/PD-L1 immune checkpoints show promising blockage effects on PD-1/PD-L1 interactions without irAEs. However, proper analytical methods and bioassays are required to effectively screen small molecule derived PD-1/PD-L1 inhibitors. Herein, we summarize the biophysical and biochemical assays currently employed for the measurements of binding capacities, molecular interactions, and blocking effects of small molecule inhibitors on PD-1/PD-L1. In addition, the discovery of natural products based PD-1/PD-L1 antagonists utilizing these screening assays are reviewed. Potential pitfalls for obtaining false leading compounds as PD-1/PD-L1 inhibitors by using certain binding bioassays are also discussed in this review.

Programmed cell death protein 1 (PD-1) and its ligand PD-L1 are critical for the regulation of T cell exhaustion and activity suppression. Tumor cells expressing immune checkpoints including PD-L1 escape monitoring of T cells from the host immune system. Checkpoint inhibitors are highly promising therapies that function as tumor-suppressing factors via modulation of tumor cell–immune cell interactions as well as boosting T cell-mediated anti-tumor immunity. Notably, PD-1 or PD-L1 monoclonal antibody (mAb) has demonstrated promising therapeutic effects in clinical studies of many types of cancer. These mAbs have caused significant tumor regression with impressive anti-tumor response rates as well as a favorable safety profile in cancer patients. Furthermore, the combination of PD-1/PD-L1 mAbs with other types of anti-tumor agents has also developed to boost the anti-tumor responses and enhance therapeutic effects in cancer patients. This review clarifies the mechanisms of PD-1/PD-L1-mediated anti-cancer immune responses and some clinical studies of mAbs targeting PD-1/PD-L1. The challenges and future of PD-1/PD-L1 blockade therapy are also discussed.

In this perspective, we discuss the current challenges and opportunities in multi-omics, a rapidly evolving approach that integrates multiple molecular layers to advance our understanding of complex biological systems. As biomedical research moves toward precision medicine, the ability to correlate genotype, phenotype, and environmental contexts has never been more critical. Multi-omics enhances biomarker discovery and elucidates regulatory networks underlying health and disease. The dominant scientific paradigm for over a century was to take a reductionist approach, studying individual molecular components in isolation or as simplified systems. The advent of omics technologies in the 1990s enabled a systems paradigm, allowing holistic analyses of molecular networks. These early systems studies were constrained by technology and methodology to bulk tissue measurements and single-omics analyses. Recent advances in single-cell and spatial omics, high-throughput proteomics and metabolomics, cloud computing, and artificial intelligence now allow high-resolution, spatially contextualized multi-omics analyses. Despite these gains, challenges in data analysis and interpretation remain, including high dimensionality, missing or incomplete data, multiple batch effects, and method-specific variability. Emerging strategies—such as paired data collection, staged or joint integration, and latent factor or quasi-mediation frameworks—offer promising solutions, positioning multi-omics as a transformative tool for elucidating complex mechanisms and guiding personalized medicine. Continued refinement of these approaches may further enhance the utility of multi-omics for understanding complex biological systems.

Introduction: Cannabinoids are reported to suppress the growth of ovarian cancer cells, but it is unclear whether structural modifications can improve their cytotoxic effects.Methods: Herein, an investigation into the antiproliferative effects of natural cannabinoids on human ovarian cancer Caov-3 cells identified cannabidiol (CBD) as the most promising cannabinoid. Furthermore, chemical modifications of CBD yielded a group of derivatives with enhanced cytotoxicity in Caov-3 cells.Results: Two CBD piperazinyl derivatives (19 and 21) showed augmented antiproliferative effects with an IC₅₀ of 5.5 and 4.1 µM, respectively, compared to CBD’s IC₅₀ of 22.9 µM. Further studies suggest that modulation of apoptosis and ferroptosis may contribute to the cytotoxic effects of CBD and its derivatives. In addition, CBD and its derivatives (19 and 21) were explored for their potential synergistic antiproliferative effects in combination with chemotherapeutic agent cisplatin. Compounds 19 or 21 (5 µM) combined with cisplatin (1 µM) showed a synergistic effect with a combination index of 0.23 and 0.72, respectively. This effect was supported by elevated levels of reactive oxygen species in Caov-3 cells treated with cisplatin combined with 19 or 21.Discussion: Findings from this study suggest that CBD derivatives with enhanced antiproliferative effects may exert synergistic effects with chemotherapeutic drugs, providing insight into the development of cannabinoid-based adjuvant agents for the management of ovarian cancer.

Precursors of advanced glycation endproducts, namely, reactive carbonyl species (RCSs), are aging biomarkers that contribute to cell death. However, the impact of RCSs on ferroptosis—an iron-dependent form of cell death—in skin cells remains unknown. Herein, we constructed a cellular model (with human keratinocyte; HaCaT cells) to evaluate the cytotoxicity of the combinations of RCSs (including glyoxal; GO and methyglyoxal; MGO) and erastin (a ferroptosis inducer) using bioassays (measuring cellular lipid peroxidation and iron content) and proteomics with sequential window acquisition of all theoretical mass spectra. Additionally, a data-independent acquisition approach was used to characterize RCSs’ and erastin’s molecular network including genes, canonical pathways, and upstream regulators. Using this model, we evaluated the cytoprotective effects of two dietary flavonoids including cannflavins A and B against RCSs and erastin-induced cytotoxicity in HaCaT cells. Cannflavins A and B (at 0.625 to 20 µM) inhibited ferroptosis by restoring the cell viability (by 56.6–78.6% and 63.8–81.1%) and suppressing cellular lipid peroxidation (by 42.3–70.2% and 28.8–63.6%), respectively. They also alleviated GO + erastin- or MGO + erastin-induced cytotoxicity by 62.2–67.6% and 56.1–69.3%, and 35.6–54.5% and 33.8–62.0%, respectively. Mechanistic studies supported that the cytoprotective effects of cannflavins A and B are associated with their antioxidant activities including free radical scavenging capacity and an inhibitory effect on glycation. This is the first study showing that cannflavins A and B protect human keratinocytes from RCSs + erastin-induced cytotoxicity, which supports their potential applications as dietary interventions for aging-related skin conditions.

The replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its main protease (M^pro), which is a plausible therapeutic target for coronavirus disease 2019 (COVID-19). Although numerous in silico studies reported the potential inhibitory effects of natural products including cannabis and cannabinoids on SARS-CoV-2 M^pro, their anti-M^pro activities are not well validated by biological experimental data. Herein, a library of minor cannabinoids belonging to several chemotypes including tetrahydrocannabinols, cannabidiols, cannabigerols, cannabichromenes, cannabinodiols, cannabicyclols, cannabinols, and cannabitriols was evaluated for their anti-M^pro activity using a biochemical assay. Additionally, the binding affinities and molecular interactions between the active cannabinoids and the M^pro protein were studied by a biophysical technique (surface plasmon resonance; SPR) and molecular docking, respectively. Cannabinoids tetrahydrocannabutol and cannabigerolic acid were the most active M^pro inhibitors (IC₅₀ = 3.62 and 14.40 μM, respectively) and cannabigerolic acid had a binding affinity $K_D=2.16\times {10}^{-4}$ M). A preliminary structure and activity relationship study revealed that the anti-${\mathrm{M}}^{\mathrm{pro}}$ effects of cannabinoids were influenced by the decarboxylation of cannabinoids and the length of cannabinoids’ alkyl side chain. Findings from the biochemical, biophysical, and computational assays support the growing evidence of cannabinoids’ inhibitory effects on SARS-CoV-2 M^pro.