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. 2025 Mar 10;16(2):101259. doi: 10.1016/j.jpha.2025.101259

Comparative two-dimensional NKG2A/CD94 cell membrane chromatography for targeted screening immune checkpoint inhibitors

Yanting Li a,b,1, Yanqiu Gu c,1, Weiyue Zhang d,1, Tianhua Li b, Chun Chen c, Chengliang Wang c, Yifeng Chai b, Xueqin Ma a,⁎⁎, Xiaofei Chen b,⁎
PMCID: PMC12966596  PMID: 41798068

Abstract

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.

Keywords: NKG2A/CD94, Immune checkpoint inhibitors, Biological chromatography, Cell membrane chromatography

Graphical abstract

Image 1

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Highlights

  • •

    A comprehensive 2D NKG2A/CD94 and HEK293 CMC comparative analysis system was successfully developed to screen for ICIs.

  • •

    Chicoric acid and alpinetin were screened as the active ingredients for NKG2A/CD94 from EP and AKH.

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    The developed comparative analysis system is suitable for other immune checkpoint inhibitors.

1. Introduction

The advent of immune checkpoint inhibitors (ICIs) has revolutionized cancer immunotherapy, providing patients with novel and efficacious therapeutic options. Monoclonal antibodies targeting programmed cell death protein 1 (PD-1) and its ligand PD-L1 have demonstrated remarkable and durable clinical responses in a subset of patients, significantly enhancing long-term oncological outcomes [1]. While these therapeutic breakthroughs have been transformative, the profound clinical responses observed are limited to a subset of patients and specific cancer types, emphasizing the pressing need to develop more robust treatment strategies that engage alternative immune checkpoint mechanisms [2].

Natural killer (NK) cells possess the intrinsic ability to recognize and selectively eradicate virally infected or neoplastic cells [3]. NK group 2 member A (NKG2A) is an immunoreceptor predominantly expressed on the surface of NK cells, with inducible expression also observed on specific T cells, particularly CD8+ cytotoxic T cells [4]. NKG2A belongs to a family of lectins, which forms a heterodimer with C-type lectin domain family 4 member A (CD94), another C-type lectin expressed by NK cells [5]. The NKG2A/CD94 complex recognizes and binds to the human leukocyte antigen (HLA)-E in humans and its murine ortholog Qa-1b, thereby transmitting inhibitory signals that effectively suppress the cytotoxic activity of both NK and CD8+ T cells [6]. Antibody-mediated blockade of NKG2A-HLA-E interactions effectively activates NK cells and potentiates immune responses against tumors, a strategy that is currently under evaluation in numerous clinical trials [7]. Thus, the study of NKG2A antibodies reveals its key role in regulating NK cell function and cancer immune surveillance. Despite their demonstrated clinical efficacy, NKG2A antibody-based therapies exhibit inherent limitations, including suboptimal oral bioavailability, prolonged tissue retention, extended half-life, and inadequate membrane permeability [8]. Small-molecule inhibitors are expected to address these challenges, providing more convenient and cost-effective therapeutic alternatives. However, to date, no small-molecule inhibitors targeting NKG2A/CD94 have been reported. Considering the potential advantages of small-molecule drugs, the development of such inhibitors would represent a valuable advancement in this field.

Traditional Chinese medicine (TCM) represents a rich repository of bioactive compounds and serves as an indispensable resource in the therapeutic management of diverse human diseases [9]. Due to its potential therapeutic efficacy and limited side effects, small molecule immune checkpoint therapy has become a significant area of global research interest. Nevertheless, numerous challenges persist in the development of small-molecule drugs targeting immune checkpoints, primarily due to a lack of efficient methods for assessing drug-immune checkpoint interactions, which significantly hinders their discovery. Recently, various biological models have been employed to screen bioactive compounds from natural products [[9], [10], [11], [12]]. Cell membrane chromatography (CMC) employs cell membranes as stationary phases to assess the binding characteristics between ligands and membrane receptors, facilitating the accurate identification of specific targets in complex samples [[13], [14], [15]]. Our group has developed a comprehensive two-dimensional (2D) CMC system and successfully screened several active components from various TCMs using different cell lines [16,17]. However, the traditional CMC suffers from some problems such as insufficient analytical throughput, short column life, and poor specificity, which prevent it from identifying the interaction receptor or carrying out drug screening for specific targets. Thus, our group developed a systematic and innovative stationary phase preparation technology based on protein biosynthesis and chemical covalent modification, as well as an innovative analysis technology based on comprehensive 2D chromatography combined with a mass spectrometry (MS) system, which significantly improved the applicability of CMC in the analysis of complex drug systems [18]. In addition, a two-channel comparative 2D analysis system was established in our previous research [19], which realized automatic switching and comparative analysis of different chromatographic columns through dual valves in series, significantly improving the efficiency and throughput of screening active ingredients.

In this study, NKG2A/CD94 was overexpressed on HEK293 cells, preserving the transmembrane structure and ligand-binding pocket of the NKG2A/CD94 complex, which successfully simulated the binding between molecules and NKG2A/CD94 in vivo. To solve the problems of insufficient bonding strength between proteins and the stationary phase, and the short lifespan of chromatographic columns, our study adopted the technology of 3-mercaptopropyltrimethoxysilane (MPTS)-N-(4-maleimide butyryl oxide) succinimide (GMBS) cross-linked stationary phase developed by our group, to gently and efficiently covalently bond cell membranes to silica gel [18]. Subsequently, a comprehensive 2D NKG2A/CD94 and HEK293 CMC comparative analysis system was developed to screen potential ICIs from Echinacea purpurea (L.) Moench (EP) and Alpinia katsumadai Hayata (AKH). Compared to previous models that focused on single-target overexpressed cells, the established comparative analysis models achieved specific and accurate screening while maintaining the biologically active conformation of NKG2A/CD94. Finally, the affinity of chicoric acid and alpinetin in the NKG2A/CD94 CMC column was higher than that in the HEK293 CMC column, suggesting they are potential NKG2A/CD94 active ingredients (Fig. 1). To validate chicoric acid and alpinetin, we conducted biophysical validation, including surface plasmon resonance (SPR) and molecular docking assays, along with NK cell activation analysis to confirm their immunomodulatory effects. By focusing on the screening of immune checkpoints, this system serves as an effective tool for integrating TCMs into cancer immunotherapy and facilitates the discovery and development of novel immunomodulatory drugs. It can also provide new ideas and technologies for accurate screening and activity evaluation of membrane receptor-targeted drugs.

Fig. 1.

Fig. 1

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Systematic flow of comparative two-dimensional (2D) natural killer (NK) group 2 member A/C-type lectin domain family 4 member A (NKG2A/CD94) and HEK293 cell membrane chromatography (CMC) for screening NK cell immune checkpoint inhibitors (ICIs). The numbers 1 and 2 indicate candidate compounds. MPTS: 3-mercaptopropyltrimethoxysilane; GMBS: N-(4-maleimide butyryl oxide) succinimide; CMSP: cell membrane stationary phase; TOF-MS: time-of-flight mass spectrometry.

2. Materials and methods

2.1. Construction of NKG2A/CD94 overexpressed HEK293 cell lines

The lentivirus encoding NKG2A/CD94 was constructed in Shanghai Jikai Gene Medical Technology Co., Ltd. (Shanghai, China). To obtain HEK293 cells with stable overexpression of NKG2A/CD94, the cells were infected with NKG2A/CD94 overexpression lentivirus in the presence of 20 μL HitransG P (Shanghai Jikai Gene Medical Technology Co., Ltd.) and subsequently selected with 2 μg/mL puromycin (Beyotime Biotechnology, Shanghai, China) for two weeks.

2.2. Preparation of the NKG2A/CD94 CMC column

The NKG2A/CD94-HEK293 and HEK293 cell membranes were prepared according to a previously established protocol [20]. Briefly, 2 × 107 NKG2A/CD94-HEK293 and HEK293 cells were harvested and subjected to disruption using an ultrasonic device (Elma, Singen, Germany) at 400 W for seven cycles, with each cycle lasting 2 s followed by a 20-s pause. The resulting cell suspension was then centrifuged at 1000 g for 10 min, and the supernatant was carefully collected. The supernatant was subsequently centrifuged again at 12,000 g for 20 min to isolate the cell membrane fraction.

To establish the NKG2A/CD94-HEK293 and HEK293 cell membrane stationary phase (CMSP), the cell membrane suspension was covalently linked to pre-dried and activated MPTS-modified silica gel (0.04 g). This process was carried out under vacuum conditions at 4 °C, with the mixture being rotated overnight for thorough binding. Following washing, the CMSP was resuspended in phosphate-buffered saline (PBS). The final CMSP suspension was packed into a column (10 mm × 2 mm i.d., 5 μm; Agilent Technologies, Santa Clara, CA, USA) employing a wet-packing procedure optimized by our team [14].

2.3. Establish of comparative 2D CMC system

Fig. S1 demonstrates that the NKG2A/CD94 CMC column/C18 column/time-of-flight (TOF)/MS system was primarily composed of an Agilent 1200 series ultra-high-performance liquid chromatography (UHPLC) system and an Agilent 6220 TOF/MS [21]. The primary column utilized for the first dimension was equipped with an NKG2A/CD94 CMC configuration. The mobile phase for this stage consisted of 10 mM ammonium acetate, delivered at a flow rate of 0.2 mL/min. For the second dimension, an XBridge TM C18 column (100 mm × 3.0 mm i.d., 3.5 μm, Waters Corporation, Wexford, Ireland), was employed. Additionally, two Acquity HSS T3 columns (2.1 mm × 50 mm i.d., 1.8 μm; Waters Corporation) were used as enrichment columns. A linear gradient elution method was employed, utilizing a mobile phase composed of 0.1 % (v/v) formic acid in aqueous solution and acetonitrile as the organic modifier, with a flow rate of 0.8 mL/min [22].

More experimental details, including reagents, instruments, cell culture, methods, sequences of NKG2A/CD94, and HLA-E are displayed in the Supplementary data.

3. Results and discussion

3.1. Characterization of NKG2A/CD94 CMC column

The expression of the NKG2A/CD94 protein was evaluated by Western blot analysis. As illustrated in Fig. 2A, the levels of NKG2A/CD94 protein were notably elevated in HEK293 cells compared to those in the control HEK293 cells, validating the successful establishment of NKG2A/CD94-HEK293 cells. Fig. 2B demonstrates that NKG2A antibody-based immunoblotting revealed the digestion of immobilized NKG2A/CD94 by radioimmunoprecipitation assay (RIPA) lysis buffer from the NKG2A/CD94/CMSP, confirming the successful covalent immobilization of NKG2A/CD94. Through scanning electron microscopy (SEM), the morphological characteristics of NKG2A/CD94/CMSP were analyzed. The cell membrane was observed to be tightly adhered to the silica support (Fig. 2C). Fluorescence microscopy was employed to assess the biological properties of NKG2A/CD94/CMSP by detecting the presence of the NKG2A/CD94 membrane protein on the silica surface. In the constructed NKG2A/CD94 plasmid, mCherry protein was fused to NKG2A. As shown in Fig. 2D, an even distribution of red fluorescence across the CMSP surface was observed, indicating that the NKG2A/CD94 protein was not only successfully expressed but also maintained its proper transmembrane structure and binding activity. In contrast, no red fluorescence from mCherry-tagged NKG2A/CD94 was detected on the HEK293 cell membrane. Consequently, the NKG2A/CD94 protein on the cell membrane was immobilized on silica, serving as a foundation for the screening of inhibitors by CMC.

Fig. 2.

Fig. 2

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Characterization of natural killer (NK) group 2 member A/C-type lectin domain family 4 member A (NKG2A/CD94) cell membrane chromatography (CMC) column. (A) Western blot analysis of NKG2A/CD94 protein expression. (B) Western blot analysis of NKG2A/CD94 protein bound to silica. (C) Electron microscopy of NKG2A/CD94 proteins on silica. (D) Fluorescence microscopy of NKG2A/CD94 protein bound to silica. (E, F) Selectivity of the NKG2A/CD94 and HEK293 CMC columns: the retention behavior of monalizumab (E) and cetuximab (F). GAPDH: glyceraldehyde 3-phosphate dehydrogenase; CMSP: cell membrane stationary phase.

The selectivity characteristics of the NKG2A/CD94 CMC system were systematically evaluated using cetuximab, an epidermal growth factor receptor (EGFR)-targeting monoclonal antibody, and monalizumab as the positive control, through chromatographic analysis. On the NKG2A/CD94 CMC column, monalizumab exhibited obvious retention, while the HEK293 CMC column showed no obvious retention of monalizumab (Fig. 2E), indicating that the NKG2A/CD94 CMC column has satisfactory specificity. As illustrated in Fig. 2F, the negative cetuximab did not show retention in the NKG2A/CD94 and HEK293 CMC columns. These results indicate that the comprehensive 2D NKG2A/CD94 and HEK293 CMC comparative analysis system is capable of screening and identifying potential components that exhibit affinity for NKG2A/CD94. The experimental findings demonstrate that the comprehensive 2D NKG2A/CD94 and HEK293 CMC comparative analysis system provides a robust system for screening and characterizing potential components with specific binding affinity toward the NKG2A/CD94 complex.

3.2. Screening anti-NKG2A/CD94 ingredients from EP and AKH

CMC serves as an efficient, stable, and highly selective method for processing complex samples, especially in the investigation of drug-receptor interactions and the screening of active compounds [[23], [24], [25]]. The comprehensive 2D NKG2A/CD94 and HEK293 CMC comparative analysis system provide an efficient and selective screening method to identify and characterize target compounds acting on NKG2A/CD94 from complex samples, which has important research implications for the development of new ICIs and the enhancement of tumor immunotherapy efficacy.

The traditional medicinal herbs EP and AKH are widely recognized as among the most frequently utilized. Flavonoids and phenolic acids, the primary active constituents in EP and AKH, exhibit significant anti-inflammatory, anticancer, and immunomodulatory effects through various mechanisms and signaling pathways [26,27]. A comprehensive 2D NKG2A/CD94 and HEK293 CMC comparative analysis system was utilized to screen potentially active compounds based on the different retention profiles of EP and AKH on the two CMC platforms. Figs. 3A–D illustrate the 2D chromatograms profiles of candidate bioactive compounds screened from EP and AKH extracts, which exhibit the capability to interact with the NKG2A/CD94 and HEK293 CMC systems. Compound identification was performed using quadrupole TOF (QTOF)/MS, with experimentally derived molecular weights systematically compared against a pre-established compound database. Additional details concerning these compounds are presented in Table S1. As shown in Figs. 3A and B, chicoric acid, compared to chlorogenic acid, showed a significantly greater binding affinity for the NKG2A/CD94 CMC column compared to the HEK293 CMC column. Similarly, from Figs. 3C and D, we can see that alpinetin, compared to kaempferol and catechin, displayed significantly stronger binding affinity for the NKG2A/CD94 CMC column, while the affinity significantly decreased on the HEK293 CMC column. These results suggest that both chicoric acid and alpinetin exhibit high affinity for NKG2A/CD94 binding. Therefore, chicoric acid and alpinetin standards were used for further verification. As shown in Figs. 3E and F, a comparison of the affinity differences between the NKG2A/CD94 and HEK293 CMC models revealed that chicoric acid and alpinetin demonstrated significantly better retention in the NKG2A/CD94 CMC column, whereas their retention in the HEK293 CMC column was notably weaker. Hence, they can be considered as specific components of NKG2A/CD94 inhibitors. The standard products of the other three compounds were also verified as shown in Fig. S2. The retention characteristics consistently correlated with the initial screening results, thereby confirming the robust molecular recognition capacity of the comprehensive 2D CMC analytical platform. Some studies have reported chicoric acid as an immune promoter and immunomodulator that stimulates phagocytosis and protects collagen III from free radicals [28]. Alpinetin demonstrates therapeutic potential for managing ulcerative colitis and related inflammatory conditions through its capacity to suppress the production of key pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) [29]. Therefore, chicoric acid and alpinetin were chosen for an in-depth investigation into their effects on NKG2A+ NK cell activation, thereby further validating the accuracy of our screening system.

Fig. 3.

Fig. 3

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Screening of affinity components of the natural killer (NK) group 2 member A/C-type lectin domain family 4 member A (NKG2A/CD94) from the Echinacea purpurea (L.) Moench (EP) and Alpinia katsumadai Hayata (AKH). (A) Typical two-dimensional (2D) plots of EP extracts obtained by the NKG2A/CD94 and HEK293 cell membrane chromatography (CMC) columns. (B) Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) spectra of chicoric acid potential active compound. (C) 2D plots of AKH extracts obtained by the NKG2A/CD94 CMC (left) and HEK293 CMC (right) columns. (D) UHPLC-QTOF/MS spectra of alpinetin potential active compound. (E) 2D plots of chicoric acid and alpinetin mixed standards generated by NKG2A/CD94 CMC (left) and HEK293 CMC (right) columns. (F) Comparison of the adjusted retention times of chicoric acid and alpinetin mixed standards in the NKG2A/CD94 and HEK293 CMC columns.

3.3. Affinity validation by SPR analysis

To further validate the direct interaction of chicoric acid and alpinetin with NKG2A/CD94, the affinity constants of chicoric acid and alpinetin were determined using the SPR assay. Serial concentrations of both compounds were assessed, resulting in affinity constants of 12.9 μM for chicoric acid (Fig. 4A) and 9.49 μM for alpinetin (Fig. 4B). These findings provide evidence that both chicoric acid and alpinetin are capable of directly binding to NKG2A/CD94. SPR analysis, widely recognized as the benchmark technique for characterizing drug-target interactions, confirmed that the bioactive candidates identified through our comprehensive 2D NKG2A/CD94 and HEK293 CMC comparative analysis system demonstrate specific and optimal binding affinity toward the recombinant protein targets. This robustly validates the efficacy of our established system in identifying target components from complex samples [30].

Fig. 4.

Fig. 4

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The interaction analysis of chicoric acid and alpinetin with natural killer (NK) group 2 member A/C-type lectin domain family 4 member A (NKG2A/CD94). (A, B) Surface plasmon resonance (SPR) sensorgrams and equilibrium dissociation constant (KD) fitting curves for chicoric acid (A) and alpinetin (B). (C, D) Molecular docking results of chicoric acid (C) and alpinetin (D).

3.4. Molecular docking assays

The analysis of binding characteristics indicated interactions between the drugs and NKG2A/CD94, however, the precise sites of interaction have yet to be elucidated. Consequently, we performed a molecular docking study to investigate the binding modes of these two compounds with NKG2A/CD94. As illustrated in Fig. 4C, chicoric acid formed three hydrogen bonds with Gln-112, Lys-164, Lys-217, one hydrophobic bond with Val-213, and two van der Waals bonds with Gln-113 and Lys-199 in the NKG2A/CD94. At the binding interface of NKG2A/CD94, alpinetin forms four hydrogen bonds with Cys-59, Tyr-126, Asn-128, and Ser-129, as well as one hydrophobic interaction with His-118 (Fig. 4D), which revealed that chicoric acid and alpinetin could bind to the amino acid residues of NKG2A/CD94 with high binding affinity values of −5.4 and −6.9 kcal/mol. Consequently, we suppose that chicoric acid and alpinetin could be potential inhibitors of NKG2A/CD94.

3.5. Pharmacological effects of the targeted components

Monalizumab exerts its therapeutic effect by inhibiting NKG2A-mediated signaling pathways, thereby stimulating the cytolytic function of both effector CD8+ T cells and NK cell populations [31]. HLA-E serves as an inhibitory ligand for the NKG2A receptor, indicating that inhibition of this interaction could potentially augment the functional activity of NK cells [31,32]. We engineered the target cell line to investigate whether the drug could block the interaction of NKG2A with HLA-E (Fig. 5A). We construed the K562-HLA-E cell line, whose expression of HLA-E was low (Figs. 5B and Fig. S3). In this experiment, K562-HLA-E serves as the experimental group overexpressing the HLA-E, whereas K562-MCS acts as the control cell line, transfected with an empty vector containing only the multiple cloning site (MCS). The K562-MCS control is utilized to rule out non-specific effects and ensure the observed results are attributable to HLA-E overexpression.

Fig. 5.

Fig. 5

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Chicoric acid and alpinetin enhanced the cytotoxicity and activity of natural killer (NK)-92MI cells. (A) Schematic diagram of the co-culture model of NK-92MI cells with K562-human leukocyte antigen (HLA)-E and K562-multiple cloning site (MCS) or K562. (B) Western blot analysis of HLA-E in K562 cells. (C–E) The cytotoxicity of NK-92MI cells against K562-HLA-E and K562-MCS cells was evaluated using a lactate dehydrogenase (LDH) assay at an effector/target (E/T) ratio of 5:1 under the treatment of monalizumab (C), chicoric acid (D), and alpinetin (E). (F) After treatment with chicoric acid and alpinetin, the aggregation area of NK-92MI cells was quantified using ImageJ. (G) K562-HLA-E or K562-MCS cells were co-cultured with NK-92MI cells, and the effects of various concentrations of chicoric acid and alpinetin were assessed at an E/T ratio of 5:1. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

We first examined whether NKG2A/CD94 inhibitors could stimulate NKG2A+ NK cells with or without HLA-E-expressing K562 cells. After treatment with NKG2A antibodies for 12 h, we monitored changes in NK cell cytolytic activity using lactate dehydrogenase (LDH) assay. As shown in Fig. 5C, overexpression of HLA-E protected K562 cells from NK-92MI-mediated cytotoxicity, while blockade of NKG2A using monalizumab markedly increased the cytotoxic efficiency of NK-92MI cells. This finding is consistent with existing literature, thereby providing additional support for the application of our developed model in drug evaluation. After treatment of NK92 cells with varying concentrations of chicoric acid and alpinetin, NK-92MI cells were subsequently co-cultured with K562 cells, and the activating effect of these compounds on NK cells was assessed using the LDH assay. As shown in Figs. 5D and E, the presence of HLA-E significantly reduced the cytotoxicity of NK-92MI cells against K562 cells compared to its absence. Treatment with chicoric acid (10 and 20 μM) and alpinetin (20 μM) significantly increased the cytotoxicity of NK-92MI cells against K562-HLA-E cells. Additionally, the addition of chicoric acid and alpinetin in these experiments did not significantly alter cell viability, suggesting that the indicated concentrations of both compounds did not induce cytotoxicity in these cells (Fig. S4).

The impact of drugs on the vitality and function of NK cells can be mediated through various mechanisms, including the facilitation of NK cell proliferation and cytotoxicity, modulation of activation and receptor expression, as well as influence on cytokine production. These factors can subsequently affect the aggregation of NK cells. The K562-HLA-E and K562 cells were co-cultured with NK-92MI cells, and different concentrations of chicoric acid and alpinetin were added to the K562-HLA-E and NK-92MI cell co-culture model for a 12 h incubation period. The vitality of NK-92MI cells was evaluated through microscopic observation of cell aggregation, where larger aggregates indicated enhanced cellular vitality. Co-culturing NK-92MI cells with K562-HLA-E cells resulted in a smaller aggregation area compared to co-culturing with K562 cells. Treatment with chicoric acid (10 and 20 μM) and alpinetin (20 μM) increased the aggregation area of NK-92MI cells. The results demonstrated that both compounds significantly enhanced the suppressive effect of K562-HLA-E cells on NK cell cytotoxicity (Figs. 5F and G).

We then investigated the activation activity of chicoric acid and alpinetin on effector cells by measuring CD107a cell surface expression in NK-92MI cells co-cultured with K562 tumor target cells expressing HLA-E. CD107a is a critical membrane marker for the degranulation process, which is essential for the cytolytic activity of NK-92MI cells. The activation of NK-92MI cells was observed in the presence of K562 cells, which do not express HLA-E. However, when HLA-E expression was induced on K562 cells, a reduction in the frequency of CD107a+ NKG2A+ NK-92MI cells was observed. When chicoric acid (20 μM) and alpinetin (20 μM) were added to the assay, the production of CD107a by NKG2A+ NK-92MI cells was restored to normal levels (Figs. 6A and B).

Fig. 6.

Fig. 6

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Chicoric acid and alpinetin unleash natural killer (NK)-92MI cell function. (A, B) NK-92MI cells were co-cultured with K562-human leukocyte antigen (HLA)-E or K562-multiple cloning site (MCS) at an effector/target (E/T) ratio of 5:1 in the presence of different concentrations of chicoric acid (A) and alpinetin (B). CD107a-+ NK-92MI cells frequencies were measured by flow cytometry. (C) Schematic diagram of the transwell co-culture model of NK-92MI and K562-MCS or K562-HLA-E. (D, E) Gene expression levels in NK-92MI cells co-cultured with K562-MCS or K562-HLA-E cells in the presence of chicoric acid (D) and alpinetin (E). APC: allophycocyanin; PE: phycoerythrin; IL-2: interleukin-2; GZMB: granzyme B; PRF: perforin; TNF-α: tumor necrosis factor-alpha; IFN-γ: interferon-gamma.

To validate the aforementioned results, a transwell co-culture system was employed to assess the impact of drugs on the activator factors in NK-92MI cells (Fig. 6C). Primer information for these factors is shown in Table S2. Quantitative real-time polymerase chain reaction (qPCR) analysis revealed that the expression levels of granzyme B (GZMB), IL-10, perforin (PRF), TNF-α, and interferon-gamma (IFN-γ) in NK-92MI cells co-cultured with HLA-E-overexpressing K562 cells was significantly diminished compared to those in HLA-E-deficient K562 cells (Fig. 6D). Treatment with chicoric acid boosted the production of GZMB, PRF, and IFN-γ in NK-92MI cells (Fig. 6D). Furthermore, the inclusion of alpinetin markedly increased PRF levels in these cells (Fig. 6E). These results suggest that the two drugs could activate NK92-MI cells, indicating their potential as promising candidates for clinical application to enhance antitumor immunity of NKG2A+ NK cells. These findings provide valuable insights for further investigation into how these drugs regulate NK cell function and may contribute to the development of novel immunotherapeutic strategies.

4. Conclusions

A novel comprehensive 2D NKG2A/CD94 and HEK293 CMC comparative analysis system was developed to identify and screen the ICIs from EP and AKH. NKG2A/CD94 overexpressed comparative analysis model maintained the natural bioactive conformation of heterodimers, which realized more accurate screening for NKG2A/CD94 inhibitors. The affinity differences of five ingredients from EP and AKH on the NKG2A/CD94 and HEK293 CMC columns were demonstrated by the system. Notably, chicoric acid and alpinetin, which exhibited the highest content and affinity, were subjected to evaluation using SPR. Results showed that they exhibited binding affinities to NKG2A/CD94 with dissociation constant (KD) values of 12.9 and 9.49 μM, respectively. It is noteworthy that both compounds exhibit not only a strong affinity for NKG2A/CD94 but also effectively bind to the contact surface of the NKG2A/CD94-spike complex, as confirmed by molecular docking studies. Furthermore, the pharmacological results indicated that chicoric acid and alpinetin may potentially modulate NK cell activation. In addition, the establishment of a membrane protein overexpression cell model within a comparative analysis system offers substantial advantages for the precise and specific screening of small-molecule drugs targeting immune checkpoints, thereby facilitating the development of novel immunotherapy strategies and drug discovery.

CRediT authorship contribution statement

Yanting Li: Writing – original draft, Visualization, Validation, Formal analysis. Yanqiu Gu: Writing – original draft, Visualization, Validation. Weiyue Zhang: Writing – original draft, Visualization, Validation. Tianhua Li: Resources. Chun Chen: Methodology, Conceptualization. Chengliang Wang: Validation. Yifeng Chai: Validation. Xueqin Ma: Writing – review & editing, Writing – original draft, Supervision, Methodology, Formal analysis, Conceptualization. Xiaofei Chen: Writing – review & editing, Writing – original draft, Supervision, Methodology, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant Nos.: 82122066, 82473884, 82073814, 82360684, and U20A20136), the National Key Research and Development Program, China (Grant No.: 2022YFC2704603), the “Dawn” program of Shanghai Education Commission, China (Grant No.: 22SG34), and the Key Research and Invention Program of Ningxia, China (Grant No.: 2023BEG02014).

Footnotes

This article is part of a special issue entitled: Targeted drug screening published in Journal of Pharmaceutical Analysis.

Peer review under responsibility of Xi'an Jiaotong University.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jpha.2025.101259.

Contributor Information

Xueqin Ma, Email: maxueqin217@126.com.

Xiaofei Chen, Email: xfchen2010@163.com.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (2.1MB, docx)

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