Improving tumor sensitivity by the introduction of an ester chain to triaryl derivatives targeting PD-1/PD-L1.

PD-1/PD-L1 pathway blockade is a promising immunotherapy for the treatment of cancer. In this manuscript, a series of triaryl compounds containing ester chains were designed and synthesized based on the pharmacophore studies of the lead BMS-1. After several SAR iterations, 22 showed the best biochemical activity binding to hPD-L1 with an IC50 of 1.21 nM in HTRF assay, and a KD value of 5.068 nM in SPR analysis. Cell-based experiments showed that 22 effectively promoted A549 cell death by restoring T-cell immune function. 22 showed significant in vivo antitumor activity in a 4T1 mouse model without obvious toxicity, with a TGI rate of 67.8 % (20 mg/kg, ip). Immunohistochemistry data indicated that 22 activates the immune activity in tumors. These results suggest that 22 is a promising compound for further development of PD-1/PD-L1 inhibitor for cancer therapy.

Development of Glycosylation-Modified DPPA-1 Compounds as Innovative PD-1/PD-L1 Blockers: Design, Synthesis, and Biological Evaluation.

In the context of peptide drug development, glycosylation plays a pivotal role. Accordingly, L-type peptides were synthesized predicated upon the PD-1/PD-L1 blocker DPPA-1. Subsequent glycosylation resulted in the production of two distinct glycopeptides, D-glu-LPPA-1 and D-gal-LPPA-1, by using D-glucose (D-glu) and D-galactose (D-gal), respectively, during glycosylation. Both glycopeptides significantly inhibited the interaction between PD-1 and PD-L1, and the measured half maximal inhibitory concentrations (IC50s) were 75.5 µM and 101.9 µM for D-glu-LPPA-1 and D-gal-LPPA-1, respectively. Furthermore, D-gal-LPPA-1 displayed a pronounced ability to restore T-cell functionality. In an MC38 tumor-bearing mouse model, D-gal-LPPA-1 demonstrated a significant inhibitory effect. Notably, D-gal-LPPA-1 substantially augmented the abundance and functionality of CD8+ T cells in the tumor microenvironment. Additionally, in the lymph nodes and spleens, D-gal-LPPA-1 significantly increased the proportion of CD8+ T cells secreting interferon-gamma (IFN-γ). These strong findings position D-gal-LPPA-1 as a potent enhancer of the antitumor immune response in MC38 tumor-bearing mice, underscoring its potential as a formidable PD-1/PD-L1 blocking agent.

Molecular hybridization used to design and synthesize neo-tanshinlactone derivatives as PD-1/PD-L1 inhibitors.

Four series of molecular hybrids (37 final products) of neo-tanshinlactone, a natural product extracted from Salvia miltiorrhiza Bunge, and known PD-1/PD-L1 interaction inhibitors were prepared as possible chemotherapeutic agents against triple negative breast cancer. Screening using a homogenous time-resolved fluorescence method resulted in three lead compounds (MZ52 IC50 74 ± 4 nM; MZ58 IC50 134 ± 17 nM; MZ61 IC50 225 ± 19 nM). With less T cell cytotoxicity and effects in activating CD8+ T cells in a T cell proliferation assay and a functionality experiment, MZ58 was selected as the best candidate for animal experiments. MZ58 exhibited antitumor effects in a subcutaneous transplantation tumor model as well as effects in reducing T cell exhaustion. In conclusion, after in vivo and in vitro experiments, we successfully acquired an effective candidate (MZ58) showing antitumor effects with low cytotoxicity toward T cells as well as the ability to activate CD8+ T cells and reduce T cell exhaustion.

Design, Synthesis, and Pharmacological Evaluation of Biaryl-Containing PD-1/PD-L1 Interaction Inhibitors Bearing a Unique Difluoromethyleneoxy Linkage.

Blockade of immune checkpoint PD-1/PD-L1 has been a promising anticancer strategy; however, clinically available PD-1/PD-L1 small-molecule inhibitors are lacking. In view of the high potency of compound 2 (BMS-1002), structural fine tuning of the methoxy linkage together with diverse modification in the solvent interaction region was conducted. A series of novel derivatives featuring a difluoromethyleneoxy linkage were designed. Compound 43 was identified as the most promising PD-1/PD-L1 inhibitor with an IC50 value of 10.2 nM in the HTRF assay. This compound is capable of promoting CD8+ T cell activation through inhibiting PD-1/PD-L1 cellular signaling. Moreover, in the Hepa1-6 syngeneic mouse model, administration of compound 43 at 1 mg/kg dosage promoted CD8+ T cell activation and delayed the tumor growth with good tolerance. Notably, the tumor in one mouse of the compound 43-treated group was completely regressed. These results indicate that compound 43 is a promising candidate worthy of further investigation.

Structure-activity relationship and biological evaluation of 12 N-substituted aloperine derivatives as PD-L1 down-regulatory agents through proteasome pathway.

Twenty-nine 12 N-substituted aloperine derivatives were synthesized and screened for suppression on PD-L1 expression in H460 cells, as a continuation of our work. Systematic structural modifications led to the identification of compound 6b as the most active PD-L1 modulator. Compound 6b could significantly down-regulate both constitutive and inductive PD-L1 expression in NSCLC cells, and successively enhance the cytotoxicity of co-cultured T cells against tumor cells at the concentration of 20 µM. Also, it exhibited a moderate in vivo anticancer efficacy against Lewis tumor xenograft with a stable PK and safety profile. The mechanism study indicated that 6b mediated the degradation of PD-L1 through a proteasome pathway, rather than a lysosome route. These results provided the powerful information for cancer immunotherapy of aloperine derivatives with unique endocyclic skeleton by targeting PD-L1 to activate immune cells to kill cancer cells.

Design, synthesis, and structure-activity relationship of PD-1/PD-L1 inhibitors with a benzo[d]isoxazole scaffold.

Blocking the programmed cell death protein 1 (PD-1) and programmed death-ligand (PD-L1) interaction has emerged as one of the most promising treatments for cancer immunotherapy. A novel series of compounds bearing a benzo[d]isoxazole scaffold was developed as PD-1/PD-L1 inhibitors, among them, compound P20 exhibited the most potent inhibitory activity, with an IC50 value of 26.8 nM. The preliminary structure-activity relationship was also investigated. The docking analysis of compound P20 with the PD-L1 dimer complex (PDB ID: 5j89) indicated that compound P20 was bound to the PD-L1 dimer with high affinity. These results suggest that compound P20 is a promising lead compound for the development of inhibitors of the PD-1/PD-L1 interaction.

Syntheses, Biological Evaluations, and Mechanistic Studies of Benzo[c][1,2,5]oxadiazole Derivatives as Potent PD-L1 Inhibitors with In Vivo Antitumor Activity.

A series of novel benzo[c][1,2,5]oxadiazole derivatives were designed, synthesized, and biologically evaluated as inhibitors of PD-L1. Among them, compound L7 exhibited 1.8 nM IC50 value in a homogeneous time-resolved fluorescence (HTRF) assay, which was 20-fold more potent than the lead compound BMS-1016. In the surface plasmon resonance (SPR) assay, L7 bound to human PD-L1 (hPD-L1) with a KD value of 3.34 nM, without showing any binding to hPD-1. In the cell-based coculture assay, L7 blocked PD-1/PD-L1 interaction with an EC50 value of 375 nM, while BMS-1016 had an EC50 value of 2075 nM. Moreover, compound L24, an ester prodrug of L7, was orally bioavailable and displayed significant antitumor effects in tumor models of syngeneic and PD-L1 humanized mice. Mechanistically, L24 exhibited significant in vivo antitumor effects probably through promoting antitumor immunity. Together, this series of benzoxadiazole PD-L1 inhibitors holds promise for tumor immunotherapy. Preclinical trials with selected compounds are ongoing in our laboratory.

Design, Synthesis, and Evaluation of o-(Biphenyl-3-ylmethoxy)nitrophenyl Derivatives as PD-1/PD-L1 Inhibitors with Potent Anticancer Efficacy In Vivo.

Two series of novel o-(biphenyl-3-ylmethoxy)nitrophenyl compounds (A1-31 and B1-17) were designed as programmed cell death protein 1 (PD-1)/PD-ligand 1 (PD-L1) inhibitors. All compounds showed significant inhibitory activity with IC50 values ranging from 2.7 to 87.4 nM except compound A17, and compound B2 displayed the best activity. Further experiments showed that B2 bound to the PD-L1 protein without obvious toxicity in Lewis lung carcinoma (LLC) cells. Furthermore, B2 significantly promoted interferon-gamma secretion in a dose-dependent manner in vitro and in vivo. Especially, B2 exhibited potent in vivo anticancer efficacy in an LLC-bearing allograft mouse model at a low dose of 5 mg/kg, which was more active than BMS-1018 (tumor growth inhibition rate: 48.5% vs 17.8%). A panel of immunohistochemistry and flow cytometry assays demonstrated that B2 effectively counteracted PD-1-induced immunosuppression in the tumor microenvironment, thereby triggering antitumor immunity. These results indicate that B2 is a promising PD-1/PD-L1 inhibitor worthy of further development.

Functional Immunostimulating DNA Materials: The Rising Stars for Cancer Immunotherapy.

Cancer immunotherapy has risen as a promising method in clinical practice for cancer treatment and DNA-based immune intervention materials, along with DNA nanotechnology, have obtained increasing importance in this field. In this review, various immunostimulating DNA materials are introduced and the mechanisms via which they exerted an immune effect are explained. Then, representative examples in which DNA is used as the leading component for anticancer applications through immune stimulation are provided and their efficacy is evaluated. Finally, the challenges for those materials in clinical applications are discussed and suggestions for possible further research directions are also put forward.

Design, synthesis, and structure-activity relationship of programmed cell death-1/programmed cell death-ligand 1 interaction inhibitors bearing a benzo[d]isothiazole scaffold.

Blockade of the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) pathway is an attractive strategy for immunotherapy. A novel series of compounds bearing a benzo[d]isothiazole scaffold were developed, among which CH20 exhibited promising activity, with an IC50 value of 8.5 nM. Further cell-based PD-1/PD-L1 blockade bioassays indicated that CH20 can inhibit the PD-1/PD-L1 interaction at the cellular level, with an EC50 value of 5.6 µM CH20 could have better potency in restoring the activity of effector cells, as the maximal luminescence values (RLUmax) of CH20 were equivalent to those of PD-L1 mAbs. The docking analysis of CH20 with the PD-L1 dimer complex (PDB ID: 6R3K) confirmed that CH20 is a promising lead compound for the development of inhibitors of the PD-1/PD-L1 interaction. The preliminary structure-activity relationship was investigated in this paper, with the aim of future drug development.

Recent advance of peptide-based molecules and nonpeptidic small-molecules modulating PD-1/PD-L1 protein-protein interaction or targeting PD-L1 protein degradation.

Tumor immunotherapy has made great progress in recent years. In the tumor microenvironment, the binding of PD-1 and its ligand PD-L1 can promote tumor immune escape and tumor survival. Clinical studies have indicated that antibodies blocking PD-1 and PD-L1 have reliable effects on many advanced malignant tumors. However, no small-molecule inhibitors have been approved so far, indicating that the development of marketable small-molecules PD-1/PD-L1 targeted therapy drugs is a challenging process. Small-molecule inhibitors can overcome the limitations of monoclonal antibodies, including poor oral bioavailability, high cost, poor tissue and tumor penetration and long half-life, which prompt researchers to turn their attention to the development of peptide molecules and small-molecule inhibitors modulating PD-1/PD-L1 to overcome some disadvantages of monoclonal antibodies or targeting PD-L1 protein degradation as potential alternatives or supplements. In this review, we will focus on the peptide-based and nonpeptidic molecules against PD-1/PD-L1 base on the structural classification. More importantly, we also focus on the latest research progress of small-molecules mediated PD-L1 degradation mechanism.

Design, synthesis, evaluation, and SAR of 4-phenylindoline derivatives, a novel class of small-molecule inhibitors of the programmed cell death-1/ programmed cell death-ligand 1 (PD-1/PD-L1) interaction.

The blockade of the PD-1/PD-L1 immune checkpoint pathway with small molecules is an emerging immunotherapeutic approach. A novel series of 4-phenylindoline derivatives were synthesized, and their inhibitory activity against the PD-1/PD-L1 protein-protein interaction (PPI) was evaluated through a homogenous time-resolved fluorescence (HTRF) assay. Among them, A20 and A22 exhibited potent activity with IC50 values of 17 nM and 12 nM, respectively. Furthermore, A20 showed the promising inhibitory activity against the PD-1/PD-L1 interaction with the EC50 value of 0.43 µM in a co-culture model of PD-L1/TCR Activator-expressing CHO cells and PD-1-expressing Jurkat cells. Besides, the structure-activity relationships (SAR) of the novel synthesized 4-phenylindoline derivatives was concluded, and the binding mode of A22 with the PD-L1 dimer was analyzed by molecular simulation and docking, demonstrating that the N-atom in the side chain of indoline fragment could interact with the amino acid residue of the PD-L1 protein to lead to the potent inhibitory activity. This study provided a new insight for further drug design.

The Stone Guest: How Does pH Affect Binding Properties of PD-1/PD-L1 Inhibitors?

The interaction between programmed cell death-1 (PD-1) and its ligand PD-L1 activates a coinhibitory signal that blocks T-cell activation, promoting the immune escape process in the tumor microenvironment. Development of monoclonal antibodies targeting and inhibiting PD-1/PD-L1 interaction as anticancer immunotherapies has proved successful in multiple clinical settings and for various types of cancer. Notwithstanding, limitations exist with the use of these biologics, including drug resistance and narrow therapeutic response rate in a majority of patients, that demand for the design of more efficacious small molecule-based immunotherapies. Alteration of pH in the tumor microenvironment is a key factor that is involved in promoting drug resistance, tumor survival and progression. In this study, we have investigated the effect of pH shifts on binding properties of distinct classes of PD-L1 inhibitors, including macrocyclic peptide and small molecules. Results expand structure-activity relationships of PD-L1 inhibitors, providing insights into structural features and physicochemical properties that are useful for the design of ligands that may escape a drug resistance mechanism associated to variable pH conditions of tumor microenvironment.

Discovery of novel CA-4 analogs as dual inhibitors of tubulin polymerization and PD-1/PD-L1 interaction for cancer treatment.

A series of novel CA-4 analogs as dual inhibitors of tubulin polymerization and PD-1/PD-L1 were designed, synthesized and bio-evaluated. Among them, compound TP5 exhibited strongest inhibitory effects against five cancer cell lines with an IC50 value of 800 nM in HepG2 cells. In addition, mechanism studies revealed that TP5 could effectively inhibit tubulin polymerization, suppress HepG2 cells migration and colony formation, and cause cell arrest at G2/M phase and induce apoptosis. Furthermore, TP5 exhibited moderate anti-PD-1/PD-L1 activity with IC50 values of 48.76 µM in a homogenous time-resolved fluorescence (HTRF) assay. In vivo efficacy studies indicated that TP5 could significantly suppress tumor growth in an immune checkpoint humanized mouse model with a Tumor Growth Suppression (TGI) of 57.9% at 100 mg/kg without causing significant toxicity. Moreover, TP5 did not cause in vivo cardiotoxicity in BALB/c mice. These results suggest that the novel CA-4 analogs may serve as a starting point for developing more potent dual inhibitors of tubulin polymerization and PD-1/PD-L1.

Covalent Organic Framework-Based Nanocomposite for Synergetic Photo-, Chemodynamic-, and Immunotherapies.

Cancer deaths are mainly caused by tumor metastases. However, tumor ablation therapies can only target the primary tumor but not inhibit tumor metastasis. Herein, a multifunctional covalent organic framework (COF)-based nanocomposite is designed for synergetic photo-, chemodynamic- and immunotherapies. Specifically, the synthesized COF possesses the ability to produce singlet oxygen under the 650 nm laser irradiation. After being metallized with FeCl3, p-phenylenediamine is polymerized on the surface of COF with Fe3+ as the oxidant. The obtained poly(p-phenylenediamine) can be used for photothermal therapy. Meanwhile, the overexpressed H2O2 in the tumor would be further catalyzed and decomposed into hydroxyl radicals (•OH) by the Fe3+/Fe2+ redox couple via Fenton reaction. Intriguingly, the increase of temperature caused by photothermal therapy can accelerate the production of •OH. Moreover, the tumor-associated antigen induced a robust antitumor immune response and effectively inhibited tumor metastasis in the presence of anti-PD-L1 checkpoint blockade. Such a COF-based multifunctional nanoplatform provides an efficacious treatment strategy for both the primary tumor and tumor metastasis.

Discovery of novel resorcinol diphenyl ether-based PROTAC-like molecules as dual inhibitors and degraders of PD-L1.

Novel resorcinol diphenyl ether-based PROTACs (PROteolysis TArgeting Chimeras) were designed and evaluated for their inhibitory activity against the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) pathway and their ability to degrade PD-L1 protein. Most of the compounds displayed excellent inhibitory activities against PD-1/PD-L1, as assessed by the homogenous time-resolved fluorescence (HTRF) binding assay, with IC50 values ranging from 25 nM to 200 nM. Among them, compound P22 is one of the best with an IC50 value of 39.2 nM. In addition to inhibiting PD-1/PD-L1 interaction, P22 also significantly restored the immunity repressed in a co-culture model of Hep3B/OS-8/hPD-L1 and CD3 T cells. Furthermore, flow cytometry (FCM) and western-blot data demonstrated that P22 could moderately reduce the protein levels of PD-L1 in a lysosome-dependent manner, which may contribute to its immune effects. Preliminary FCM and western-blot data suggest that it is possible to build PD-L1-targeting PROTAC-like molecules based on PD-1/PD-L1 small molecule inhibitors, though these compounds showed only modest degradation efficiencies. Collectively, this work suggests that P22 may serve as a starting point for exploring the degradation of PD-L1 by PROTAC-like strategy.

Preparation of Biphenyl-Conjugated Bromotyrosine for Inhibition of PD-1/PD-L1 Immune Checkpoint Interactions.

Cancer immunotherapy has been revolutionized by the development of monoclonal antibodies (mAbs) that inhibit interactions between immune checkpoint molecules, such as programmed cell-death 1 (PD-1), and its ligand PD-L1. However, mAb-based drugs have some drawbacks, including poor tumor penetration and high production costs, which could potentially be overcome by small molecule drugs. BMS-8, one of the potent small molecule drugs, induces homodimerization of PD-L1, thereby inhibiting its binding to PD-1. Our assay system revealed that BMS-8 inhibited the PD-1/PD-L1 interaction with IC50 of 7.2 µM. To improve the IC50 value, we designed and synthesized a small molecule based on the molecular structure of BMS-8 by in silico simulation. As a result, we successfully prepared a biphenyl-conjugated bromotyrosine (X) with IC50 of 1.5 µM, which was about five times improved from BMS-8. We further prepared amino acid conjugates of X (amino-X), to elucidate a correlation between the docking modes of the amino-Xs and IC50 values. The results suggested that the displacement of amino-Xs from the BMS-8 in the pocket of PD-L1 homodimer correlated with IC50 values. This observation provides us a further insight how to derivatize X for better inhibitory effect.