Background

Cancer is a global health burden resulting in high healthcare costs. Therefore, the search for effective therapeutics is of continued scientific interest. Recently, cancer immunotherapy, a strategy that utilizes the host’s own immune system to fight tumors, has become an effective treatment of cancers (Makuku et al., 2021). In cancer, the tumor cell microenvironment acts to inhibit immune checkpoints, which normally function to prevent uncontrolled proliferation (He and Xu, 2020). Programmed cell death protein 1 (PD-1), an immune checkpoint expressed by several types of immune cells, dampens the immune system upon programmed cell death ligand-1 (PD-L1) binding (Makuku et al., 2021). The interaction of PD-1/PD-L1 leads to the inhibition of phosphorylation of the T-cell-receptor (TCR) signaling intermediate, which terminates the TCR signaling cascade (Keir et al., 2008; Fife et al., 2009). Two signaling motifs in the cytoplasmic tail of PD-1 are the intracellular immunoreceptor tyrosine-based switch motif (ITSM) and the immunoreceptor tyrosine-based inhibitory motif. Upon PD-L1 binding to PD-1, ITSM is phosphorylated and recruits Src homology 2-containing tyrosine phosphatase, thereby inhibiting the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway (Barclay et al., 2018; Ai et al., 2020). PI3K/Akt signaling pathway blockage further downregulates the mechanistic targets of rapamycin and inhibits protein synthesis and cell growth. PI3K/Akt signaling pathway blockage also inhibits the degradation of transcription factor FoxO1, which enhances the expression of PD-1 (Barclay et al., 2018; Ai et al., 2020).

The recognition of the PD-1 protein on the membrane of T cells by tumor cells results in the upregulation of PD-L1 (J. Liu et al., 2021). High expression of PD-L1 is one characteristic observed in many types of tumors including melanoma, lung cancer, and breast cancer (Mu et al., 2011; Fusi et al., 2015; Aguilar et al., 2019). PD-1/PD-L1 binding results in T-cell apoptosis (J. Liu et al., 2021). Blockade of the PD-1/PD-L1 axis results in tumor suppression due to interference between the tumor cell and the T cell (C. Liu et al., 2021; Makuku et al., 2021). Numerous studies have demonstrated that the blockage of PD-L1 or PD-1 is one of the most promising approaches for cancer immunotherapy (Zitvogel and Kroemer, 2012; Wu et al., 2018; Salmaninejad et al., 2019). Blocking the interactions of PD-L1 and PD-1 shuts off the inhibitory signaling pathways for T cells, reactivates the T cell–mediated anti-tumor responses by promoting T-cell proliferation, and enhances effector T-cell function (Salmaninejad et al., 2019; Tang and Zheng., 2018). Clinical data have demonstrated that the blockade of PD-1 or PD-L1 can boost T cell–mediated antitumor responses, generates durable clinical responses, and prolongs patient survival time (Ohaegbulam et al., 2015; Alsaab et al., 2017). Monoclonal antibodies against PD-1 (Pembrolizumab, Nivolumab, and Cemiplimab) or PD-L1 (Atezolizumab, Avelumab, and Durvalumab) have been approved by FDA for the treatment of a series of malignancies including breast cancer, bladder cancer, colorectal cancer, lung cancer, hepatoma, and melanoma (Massard et al., 2016; Kim, 2017; Xin Yu et al., 2020). Although these monoclonal antibodies demonstrated promising results with high clinical efficacy and immune-related adverse effects, immunogenicity and high costs are still major limitations of antibody-based immune checkpoint inhibitors (Hamanishi et al., 2015; Alsaab et al., 2017; Zinzani et al., 2017; Akinleye and Rasool et al., 2019). Alternatively, small-molecule inhibitors can overcome these advantages due to better tumor penetration and oral availability (Zhan et al., 2016).

Therefore, the discovery of small molecule inhibitors blocking the PD-1/PD-L1 interaction is a promising cancer therapy approach. Our group reported the evaluation of the PD-1/PD-L1 blockade (using a pair-ELISA technique) and the binding of compounds to either PD-1 or PD-L1 (Li et al., 2022). However, this method is not efficient for large-scale screenings of small molecule libraries for PD-1/PD-L1 inhibitors. Therefore, we developed a surface plasmon resonance (SPR)-based PD-1/PD-L1 blockade screening approach utilizing immobilized PD-1 (on the chip), PD-L1 (in solution), and known inhibitors (i.e., BMS-1166 or BMS-202, in solution). To exclude potential false positives, we included a negative PD-L1 inhibitor (NO-Losartan A) possessing a biphenyl group—a structural feature shared with the BMS-1166 and the BMS-202 compounds that were investigated together in this study. Notably, the SPR technique is a valuable complementary method to ELISA immunoassays that can also be used for the optimization of ELISA-based assays (Vaisocherová et al., 2009). SPR is an optical biosensor technology that employs the evanescent wave phenomenon to detect changes in the refractive index of a biosensor (Pattnaik, 2005). A light source illuminates the biosensor and prism, and as the analyte flows through the channel and binds to the target protein, the refractive index of the biosensor undergoes a shift. This interaction between analyte and protein is monitored in real-time, enabling precise measurement of the amount of bound protein as well as the rates of association and dissociation. The SPR assay has unique advantages over the ELISA-type assay. Rather than merely providing an endpoint, the SPR assay monitors the kinetics associated with the PD-1/PD-L1 blockade of small molecules in real time. We acknowledge that ELISA-type assays are more widely accessible and adaptable to different laboratory settings, and we recognize that our SPR assay requires specialized instrumentation and expertise, which may not be available in all laboratories. Furthermore, given that this blockade assay is solely based on in vitro experiments, it is imperative to perform functional assays and in vivo validation to confirm the potential of compounds exhibiting blockade effects against PD-1/PD-L1. However, we believe that this SPR-based protocol, which provides sufficient details, can facilitate the screening process of small molecule inhibitors that block the PD-1/PD-L1 interaction at a large scale.

In the present study, we utilized an SPR-based assay to determine the IC₅₀ values of BMS-1166 and BMS-202, which were measured at 85.4 and 654.4 nM, respectively. BMS-1166 has been previously characterized with an IC₅₀ value of 1.4 and 276 nM by homogeneous time-resolved fluorescence (HTRF) and cell-based assays (Jurkat cells expressing PD-1 in co-culture with CHO cells expressing PD-L1), respectively (Guzik et al., 2017). Previous investigations have reported IC₅₀ values of BMS-202 at 18 and 96 nM utilizing different assays, including cell-based and HTRF approaches (Surmiak et al., 2021). Our results are comparable with previous findings and confirm the reliability and reproducibility of our SPR-based protocol.