Discovery of novel thiophene[3,2-d]pyrimidine-based tubulin inhibitors with enhanced antitumor efficacy for combined use with anti-pd-l1 immunotherapy in melanoma.

Herein, we designed and synthesized a series of novel 2-methylthieno [3,2-d]pyrimidine analogues as tubulin inhibitors with antiproliferative activities at low nanomolar levels. Among them, compound DPP-21 displayed the most potent anti-proliferative activity against six cancer cell lines with an average IC50 of ∼6.23 nM, better than that of colchicine (IC50 = 9.26 nM). DPP-21 exerted its anti-cancer activity by suppressing the polymerization of tubulin with an IC50 of 2.4 µM. Furthermore, the crystal structure of DPP-21 in complex with tubulin was solved by X-ray crystallography to 2.94 Å resolution, confirming the direct binding of DPP-21 to the colchicine site. Moreover, DPP-21 arrested the cell cycle in the G2/M phase of mitosis, subsequently inducing tumor cell apoptosis. Additionally, DPP-21 was able to effectively inhibit the migration of cancer cells. Besides, DPP-21 exhibited significant in vivo anti-tumor efficacy in a B16-F10 melanoma tumor model with a TGI of 63.3 % (7 mg/kg) by intraperitoneal (i.p.) injection. Notably, the combination of DPP-21 with NP-19 (a PD-L1-targeting small molecule inhibitor reported by our group before) demonstrated enhanced anti-cancer efficacy in vivo. These results suggest that DPP-21 is a promising lead compound deserving further investigation as a potential anti-cancer agent.

Design, synthesis, and optimization of novel PD-L1 inhibitors and the identification of a highly potent and orally bioavailable PD-L1 inhibitor.

In this paper we report the discovery of structurally novel and highly potent programmed cell death-ligand 1 (PD-L1) inhibitors targeting surface and intracellular PD-L1. A ring fusion design utilizing dimethoxyphenyl indazole derivatives was used, followed by structural extension, which further improved potency by inducing the formation of additional symmetrical interactions within the PD-L1 binding site, leading to the discovery of novel and highly active tetra-aryl-scaffold inhibitors. Key optimizations involved polar tail chain modifications that improve potency and minimize cell cytotoxicity. In addition, druggability issues that exist outside the rule-of-five chemical space were addressed. CB31, a representative compound, was found to exhibit outstanding activity in blocking programmed cell death-1 (PD-1)/PD-L1 interactions (IC50 = 0.2 nM) and enhancing T-cell functions, with minimal cell cytotoxicity. CB31 also displayed favorable oral pharmacokinetic properties, consistent with its high passive permeability and insusceptibility to efflux transporters, as well as its high metabolic stability. Additionally, CB31 demonstrated mechanistically differentiated features from monoclonal antibodies by inducing PD-L1 internalization, intracellular retention of PD-L1 with altered glycosylation pattern, and PD-L1 degradation. It also demonstrated greater effects on tumor size reduction and tumor cell killing, with enhanced T-cell infiltration, in a 3D tumor spheroid model. Overall, results show that CB31 is a promising small-molecule PD-L1 inhibitor that can inhibit PD-1/PD-L1 interactions and promote PD-L1 degradation.

Design, synthesis, and evaluation of antitumor activity of 2-arylmethoxy-4-(2-fluoromethyl-biphenyl-3-ylmethoxy) benzylamine derivatives as PD-1/PD-l1 inhibitors.

A series of novel 2-arylmethoxy-4-(2-fluoromethyl-biphenyl-3-ylmethoxy) benzylamine derivatives was designed, synthesized, and evaluated for their antitumor effects as PD-1/PD-L1 inhibitors both in vitro and in vivo. Firstly, the ability of these compounds to block the PD-1/PD-L1 immune checkpoint was assessed using the homogeneous time-resolved fluorescence (HTRF) assay. Two of the compounds can strongly block the PD-1/PD-L1 interaction, with IC50 values of less than 10 nM, notably, compound HD10 exhibited significant clinical potential by inhibiting the PD-1/PD-L1 interaction with an IC50 value of 3.1 nM. Further microscale thermophoresis (MST) analysis demonstrated that HD10 had strong interaction with PD-L1 protein. Co-crystal structure (2.7 Å) analysis of HD10 in complex with the PD-L1 protein revealed a strong affinity between the compound and the target PD-L1 dimer. This provides a solid theoretical basis for further in vitro and in vivo studies. Next, a typical cell-based experiment demonstrated that HD10 could remarkably prevent the interaction of hPD-1 293 T cells from human recombinant PD-L1 protein, effectively restoring T cell function, and promoting IFN-γ secretion in a dose-dependent manner. Moreover, HD10 was effective in suppressing tumor growth (TGI = 57.31 %) in a PD-1/PD-L1 humanized mouse model without obvious toxicity. Flow cytometry, qPCR, and immunohistochemistry data suggested that HD10 inhibits tumor growth by activating the immune system in vivo. Based on these results, it seems likely that HD10 is a promising clinical candidate that should be further investigated.

Identification of novel PD-1/PD-L1 small molecule inhibitors: virtual screening, synthesis and in vitro characterisation.

The PD-1/PD-L1 pathway is considered as one of the most promising immune checkpoints in tumour immunotherapy. However, researchers are faced with the inherent limitations of antibodies, driving them to pursue PD-L1 small molecule inhibitors. Virtual screening followed by experimental validation is a proven approach to discover active compounds. In this study, we employed multistage virtual screening methods to screen multiple compound databases to predict new PD-1/PD-L1 ligands. 35 compounds were proposed by combined analysis of fitness scores, interaction pattern and MM-GBSA binding affinities. Enzymatic assay confirmed that 10 out of 35 ligands were potential PD-L1 inhibitors, with inhibitory rate higher than 50% at the concentration of 30 µM. Among them, ZDS20 was identified as the most effective inhibitor with low micromolar activity (IC50 = 3.27 µM). Altogether, ZDS20 carrying novel scaffold was identified and could serve as a lead for the development of new classes of PD-L1 inhibitors.

Discovery of Novel Proteolysis-Targeting Chimera Molecules as Degraders of Programmed Cell Death-Ligand 1 for Breast Cancer Therapy.

The immune checkpoint blockade represents a pivotal strategy for tumor immunotherapy. At present, various programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) monoclonal antibodies have been successfully applied to tumor treatment. Additionally, numerous small molecule inhibitors of the PD-1/PD-L1 interaction have also been developed, with some advancing into clinical trials. Here, a novel PD-L1 proteolysis-targeting chimera (PROTAC) library was designed and synthesized utilizing the PD-L1 inhibitor BMS202 and the E3 ligand PG as foundational components. Among these, we identified a highly potent molecule PA8 for PD-L1 degradation in 4T1 cells (DC50 = 0.609 µM). Significantly, compound PA8 potentially inhibits 4T1 cell growth both in vitro and in vivo. Further mechanistic studies revealed that PA8 effectively promoted the immune activation of model mice. Thus, these results suggest that PA8 could be a novel strategy for cancer immunotherapy in the 4T1 tumor model. Although PA8 exhibits weaker degradation activity in some human cancer cells, it still provides a certain basis for further research on PD-L1 PROTAC.

Design, synthesis and biological evaluation of novel quinazoline derivatives as immune checkpoint inhibitors.

In this work, we report 14 novel quinazoline derivatives as immune checkpoint inhibitors, IDO1 and PD-L1. The antitumor screening of synthesized compounds on ovarian cancer cells indicated that compound V-d and V-l showed the most activity with IC50 values of about 5 µM. Intriguingly, compound V-d emerges as a stand out, triggering cell death through caspase-dependent and caspase-independent manners. More importantly, V-d presents its ability to hinder tumor sphere formation and re-sensitized cisplatin-resistant A2780 cells to cisplatin treatment. These findings suggest that compound V-d emerges as a promising lead candidate for the future development of immuno anticancer agents.

Discovery of Novel Small-Molecule-Based Potential PD-L1/EGFR Dual Inhibitors with High Druggability for Glioblastoma Immunotherapy.

Based on the close relationship between programmed death protein ligand 1 (PD-L1) and epidermal growth factor receptor (EGFR) in glioblastoma (GBM), we designed and synthesized a series of small molecules as potential dual inhibitors of EGFR and PD-L1. Among them, compound EP26 exhibited the highest inhibitory activity against EGFR (IC50 = 37.5 nM) and PD-1/PD-L1 interaction (IC50 = 1.77 µM). In addition, EP26 displayed superior in vitro antiproliferative activities and in vitro immunomodulatory effects by promoting U87MG cell death in a U87MG/Jurkat cell coculture model. Furthermore, EP26 possessed favorable pharmacokinetic properties (F = 22%) and inhibited tumor growth (TGI = 92.0%) in a GBM mouse model more effectively than Gefitinib (77.2%) and NP19 (82.8%). Moreover, EP26 increased CD4+ cells and CD8+ cells in tumor microenvironment. Collectively, these results suggest that EP26 represents the first small-molecule-based PD-L1/EGFR dual inhibitor deserving further investigation as an immunomodulating agent for cancer treatment.

Discovery of Small and Bifunctional Molecules Targeting PD-L1/CD73 for Cancer Dual Immunotherapy.

In this work, a series of bifunctional PD-L1/CD73 (cluster of differentiation 73) small-molecule inhibitors were designed and synthesized. Among them, CC-5 showed the strongest PD-L1 inhibitory effects with an IC50 of 6 nM and potent anti-CD73 activity with an IC50 of 0.773 µM. The high PD-L1/CD73 inhibitory activity of CC-5 was further confirmed by SPR assays with KD of 182 nM for human PD-L1 and 101 nM for CD73, respectively. Importantly, CC-5 significantly suppressed tumor growth in a CT26 and B16-F10 tumor model with TGI of 64.3% and 39.6%, respectively. Immunohistochemical (IHC) and flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) indicated that CC-5 exerted anticancer effects via activating the tumor immune microenvironment. Collectively, CC-5 represents the first dual PD-L1/CD73 inhibitor worthy of further research as a bifunctional immunotherapeutic agent.

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.

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.

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.

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.

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.