Based on polydopamine-coated metal organic framework multifunctional nanoplatform for enhanced photothermal/sonodynamicand treatment combined with checkpoint blockade therapy.

To overcome the low efficacy of sonodynamic therapy (SDT) caused by hypoxia in the tumor microenvironment, we developed a multiple anti-tumor nanoplatform with synergistic SDT, photothermal therapy (PTT), and ferroptosis effects. PCN-224@FcCaO2/Mn/dihydroartemisinin/imiquimod/PDA (PFC) was prepared by modified with dihydroartemisinin (DHA), imiquimod (R837), CaO2, ferrocene (Fc) and Mn2+ on the PCN-224 (Cu) to achieve self-replenishment of H2O2/O2 and GSH consumption. FcCaO2 decomposed into H2O2 in the tumor microenvironment, triggering the Fenton effect to produce OH, and Cu2+ reduced the potential loss of OH by the depletion of GSH. Under ultrasonic (US) and laser irradiation, PFC exhibits exciting PTT and SDT effects from polydopamine (PDA) and PCN-224. Mn2+ not only promoted the reaction of H2O2 to produce O2 to effectively enhance SDT but also induced tumor cell apoptosis by Mn2+ combined with DHA. PFC induced ferroptosis via Fe interaction with DHA to produce ROS and reduce the expression of GPX4. The released R837 and tumor-associated antigens from SDT/PTT can produce damage associated molecular patterns (DAMPs), which can initiate adaptive immune responses to kill cancer cells, and released again to promote the tumor immune cycle. What's more, SDT/PTT and ferroptosis combined with aPD-L1 can effectively suppress both primary and distant tumor growth.

In Search of Better Peptide-(Derived from PD-L2)-Based Immune Checkpoint Inhibitors.

Current anti-cancer immune checkpoint therapy relies on antibodies that primarily target the PD-1/PD-L1(-L2) negative regulatory pathway. Although very successful in some cases for certain cancers, these antibodies do not help most patients who, presumably, should benefit from this type of therapy. Therefore, an unmet clinical need for novel, more effective drugs targeting immune checkpoints remains. We have developed a series of high-potency peptide inhibitors interfering with PD-1/PD-L1(-L2) protein-protein interaction. Our best peptide inhibitors are 12 and 14 amino acids long and show sub-micromolar IC50 inhibitory activity in the in vitro assay. The positioning of the peptides within the PD-1 binding site is explored by extensive modeling. It is further supported by 2D NMR studies of PD-1/peptide complexes. These results reflect substantial progress in the development of immune checkpoint inhibitors using peptidomimetics.

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.

A Redox-Triggered Autophagy-Induced Nanoplatform with PD-L1 Inhibition for Enhancing Combined Chemo-Immunotherapy.

Epirubicin (EPI) alone can trigger mildly protective autophagy in residual tumor cells, resulting in an immunosuppressive microenvironment. This accelerates the recurrence of residual tumors and leads to antiprogrammed death ligand 1 (anti-PD-1)/PD-L1 therapy resistance, posing a significant clinical challenge in tumor immunotherapy. The combination of checkpoint inhibitors targeting the PD-1/PD-L1 pathway and amplifying autophagy presents an innovative approach to tumor treatment, which can prevent tumor immune escape and enhance therapeutic recognition. Herein, we aimed to synthesize a redox-triggered autophagy-induced nanoplatform with SA&EA-induced PD-L1 inhibition. The hyaluronic acid (HA) skeleton and arginine segment promoted active nanoplatform targeting, cell uptake, and penetration. The PLGLAG peptide was cleaved by overexpressing matrix metalloproteinase-2 (MMP-2) in the tumor microenvironment, and the PD-L1 inhibitor D-PPA was released to inhibit tumor immune escape. The intense autophagy inducers, STF-62247 and EPI, were released owing to the cleavage of disulfide bonds influenced by the high glutathione (GSH) concentration in tumor cells. The combination of EPI and STF induced apoptosis and autophagic cell death, effectively eliminating a majority of tumor cells. This indicated that the SA&EA nanoplatform has better therapeutic efficacy than the single STF@AHMPP and EPI@AHMPTP groups. This research provided a way to set up a redox-triggered autophagy-induced nanoplatform with PD-L1 inhibition to enhance chemo-immunotherapy.

Reducing PD-L1 Expression by Degraders and Downregulators as a Novel Strategy to Target the PD-1/PD-L1 Pathway.

Targeting the programmed cell death protein-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) pathway has evolved into one of the most promising strategies for tumor immunotherapy. Thus far, multiple monoclonal antibody drugs have been approved for treating a variety of tumors, while the development of small-molecule PD-1/PD-L1 inhibitors has lagged far behind, with only a few small-molecule inhibitors entering clinical trials. In addition to antibody drugs and small-molecule inhibitors, reducing the expression levels of PD-L1 has attracted extensive research interest as another promising strategy to target the PD-1/PD-L1 pathway. Herein, we analyze the structures and mechanisms of molecules that reduce PD-L1 expression and classify them as degraders and downregulators according to whether they directly bind to PD-L1. Moreover, we discuss the potential prospects for developing PD-L1-targeting drugs based on these molecules. It is hoped that this perspective will provide profound insights into the discovery of potent antitumor immunity drugs.

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.

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.

Boosting Checkpoint Immunotherapy with Biomimetic Nanodrug Delivery Systems.

Immune checkpoint blockade (ICB) has achieved unprecedented progress in tumor immunotherapy by blocking specific immune checkpoint molecules. However, the high biodistribution of the drug prevents it from specifically targeting tumor tissues, leading to immune-related adverse events. Biomimetic nanodrug delivery systems (BNDSs) readily applicable to ICB therapy have been widely developed at the preclinical stage to avoid immune-related adverse events. By exploiting or mimicking complex biological structures, the constructed BNDS as a novel drug delivery system has good biocompatibility and certain tumor-targeting properties. Herein, the latest findings regarding the aforementioned therapies associated with ICB therapy are highlighted. Simultaneously, prospective bioinspired engineering strategies can be designed to overcome the four-level barriers to drug entry into lesion sites. In future clinical translation, BNDS-based ICB combination therapy represents a promising avenue for cancer treatment.

Cancer Cell-Selective PD-L1 Inhibition via a DNA Safety Catch to Enhance Immunotherapy Specificity.

Immune checkpoint protein blockade (ICB) has emerged as a powerful immunotherapy approach, but suppressing immune-related adverse events (irAEs) for noncancerous cells and normal tissues remains challenging. Activatable ICB has been developed with tumor microenvironment highly-expressed molecules as stimuli, but they still lack precision and efficiency considering the diffusion of stimuli molecules in whole tumor tissue. Here we assemble PD-L1 with a duplex DNA strand, termed as "safety catch", to regulate its accessibility for ICB. The safety catch remains at "on" status for noncancerous cells to prevent ICB binding to PD-L1. Cancer cell membrane protein c-Met acts as a trigger protein to react with safety catch, which selectively exposes its hybridization region for ICB reagent. The ICB reagent is a retractable DNA nanostring with repeating hairpin-structural units, whose contraction drives PD-L1 clustering with endocytosis-guided degradation. The safety catch, even remained at "safety on" status, is removed from the cell membrane via a DNA strand displacement reaction to minimize its influence on noncancerous cells. This strategy demonstrates selective and potent immunotherapeutic capabilities only against cancer cells both in vitro and in vivo, and shows effective suppression of irAEs in normal tissues, therefore would become a promising approach for precise immunotherapy in mice.

A multitier virtual screening of antagonists targeting PD-1/PD-L1 interface for the management of triple-negative breast cancer.

Immunotherapies are promising therapeutic options for the management of triple-negative breast cancer because of its high mutation rate and genomic instability. Of note, the blockade of the immune checkpoint protein PD-1 and its ligand PD-L1 has been proven to be an efficient and potent strategy to combat triple-negative breast cancer. To date, various anti-PD-1/anti-PD-L1 antibodies have been approved. However, the intrinsic constraints of these therapeutic antibodies significantly limit their application, making small molecules a potentially significant option for PD-1/PD-L1 inhibition. In light of this, the current study aims to use a high-throughput virtual screening technique to identify potential repurposed candidates as PD-L1 inhibitors. Thus, the present study explored binding efficiency of 2509 FDA-approved compounds retrieved from the drug bank database against PD-L1 protein. The binding affinity of the compounds was determined using the glide XP docking programme. Furthermore, prime-MM/GBSA, DFT calculations, and RF score were used to precisely re-score the binding free energy of the docked complexes. In addition, the ADME and toxicity profiles for the lead compounds were also examined to address PK/PD characteristics. Altogether, the screening process identified three molecules, namely DB01238, DB06016 and DB01167 as potential therapeutics for the PD-L1 protein. To conclude, a molecular dynamic simulation of 100 ns was run to characterise the stability and inhibitory action of the three lead compounds. The results from the simulation study confirm the robust structural and thermodynamic stability of DB01238 than other investigated molecules. Thus, our findings hypothesize that DB01238 could serve as potential PD-L1 inhibitor in the near future for triple-negative breast cancer patients.

Aptamers Targeting the PD-1/PD-L1 Axis: A Perspective.

Aptamers have emerged in recent years as alternatives to antibodies or small molecules to interfere with the immune check points by blocking the PD-1/PD-L1 interactions and represent an interesting perspective for immuno-oncology. Aptamers are RNA or DNA nucleotides able to bind to a target with high affinity, with the target ranging from small molecules to proteins and up to cells. Aptamers are identified by the SELEX method that can be modified for specific purposes. The range of applications of aptamers covers therapy as well as new alternative assay technologies similar to ELISA. Aptamers' limited plasma stability can be managed using delivery strategies. The goal of this Perspective is to give an overview of the current development of aptamers targeting the most studied immune checkpoint modulators, PD-1 and PD-L1, and analogous strategies with aptamers for other immuno-related targets.

Identification of CBPA as a New Inhibitor of PD-1/PD-L1 Interaction.

Targeting of the PD-1/PD-L1 immunologic checkpoint is believed to have provided a real breakthrough in the field of cancer therapy in recent years. Due to the intrinsic limitations of antibodies, the discovery of small-molecule inhibitors blocking PD-1/PD-L1 interaction has gradually opened valuable new avenues in the past decades. In an effort to discover new PD-L1 small molecular inhibitors, we carried out a structure-based virtual screening strategy to rapidly identify the candidate compounds. Ultimately, CBPA was identified as a PD-L1 inhibitor with a KD value at the micromolar level. It exhibited effective PD-1/PD-L1 blocking activity and T-cell-reinvigoration potency in cell-based assays. CBPA could dose-dependently elevate secretion levels of IFN-γ and TNF-α in primary CD4+ T cells in vitro. Notably, CBPA exhibited significant in vivo antitumor efficacy in two different mouse tumor models (a MC38 colon adenocarcinoma model and a melanoma B16F10 tumor model) without the induction of observable liver or renal toxicity. Moreover, analyses of the CBPA-treated mice further showed remarkably increased levels of tumor-infiltrating CD4+ and CD8+ T cells and cytokine secretion in the tumor microenvironment. A molecular docking study suggested that CBPA embedded relatively well into the hydrophobic cleft formed by dimeric PD-L1, occluding the PD-1 interaction surface of PD-L1. This study suggests that CBPA could work as a hit compound for the further design of potent inhibitors targeting the PD-1/PD-L1 pathway in cancer immunotherapy.

Validation of an LC-MS/MS method for the determination of sotorasib, a KRASG12C inhibitor, in human plasma.

Background: Sotorasib (AMG 510) is a first-in-class KRASG12C inhibitor that received accelerated US FDA approval in 2021 for the treatment of patients with KRASG12C-mutated locally advanced or metastatic non-small-cell lung cancer. Method: An LC-MS/MS method was developed and validated for the determination of sotorasib in human plasma to support clinical development studies. Samples were prepared using protein precipitation and analyzed by LC-MS/MS using gradient elution with a calibration standard curve range of 10.0-10,000 ng/ml. Stable isotope labeled [13C, D3]-sotorasib was used as an internal standard. Results & conclusion: The method fully met FDA guidelines for all validation parameters, including precision, accuracy, selectivity, matrix effect, recovery and stability and has been extensively used to support multiple clinical studies.

Molecular basis of PD-1 blockade by dostarlimab, the FDA-approved antibody for cancer immunotherapy.

Targeting of programmed cell death 1 (PD-1) with monoclonal antibodies to block the interaction with its ligand PD-L1 has been successful in immunotherapy of multiple types of cancer, and their mechanism involves the restoration of the T-cell immune response. April 2021, the US FDA approved dostarlimab, a therapeutic antibody against PD-1, for the treatment of endometrial cancer. Here, we report the crystal structure of the extracellular domain of PD-1 in complex with the dostarlimab Fab at the resolution of 1.53 Å. Although the interaction between PD-1 and dostarlimab involves mainly the residues within the heavy chain of dostarlimab, the steric occlusion of PD-L1 binding is primarily contributed by the light chain. Dostarlimab induces conformational rearrangements of the BC, C'D and FG loops of PD-1 to achieve a high affinity. Significantly, the residue R86 within the C'D loop of PD-1 plays a critical role for dostarlimab binding by occupying the concave surface on the heavy chain via multiple interactions. This high-resolution structure can provide helpful information for designing improved anti-PD-1 biologics or effective combination strategies for cancer immunotherapy.

Evaluation of Substituted Pyrazole-Based Kinase Inhibitors in One Decade (2011-2020): Current Status and Future Prospects.

Pyrazole has been recognized as a pharmacologically important privileged scaffold whose derivatives produce almost all types of pharmacological activities and have attracted much attention in the last decades. Of the various pyrazole derivatives reported as potential therapeutic agents, this article focuses on pyrazole-based kinase inhibitors. Pyrazole-possessing kinase inhibitors play a crucial role in various disease areas, especially in many cancer types such as lymphoma, breast cancer, melanoma, cervical cancer, and others in addition to inflammation and neurodegenerative disorders. In this article, we reviewed the structural and biological characteristics of the pyrazole derivatives recently reported as kinase inhibitors and classified them according to their target kinases in a chronological order. We reviewed the reports including pyrazole derivatives as kinase inhibitors published during the past decade (2011-2020).

PTCH1 mutation promotes antitumor immunity and the response to immune checkpoint inhibitors in colorectal cancer patients.

Immunotherapy has emerged as an effective therapeutic strategy for various cancers, including colorectal cancer (CRC), but only a subset of MSI-H patients can benefit from such therapy. Patched1 (PTCH1) is a frequently altered gene in CRCs and its mutations contribute to unregulated Hedgehog (Hh) signaling. In the study, we evaluated the association of PTCH1 mutations with CRC immunity based on our single-center cohort and multiple cancer genomic datasets. Among 21 enrolled patients, six (28.6%) harbored a PTCH1 mutation based on WES analyses. In CRC patients, the PTCH1 mutation subgroup experienced a higher durable clinical benefit rate than the PTCH1 wild-type subgroup (100% vs. 40%, P = 0.017). In addition, patients with the PTCH1 mutation experienced greater progression-free survival (PFS, P = 0.037; HR, 0.208) and overall survival (OS, P = 0.045; HR, 0.185). A validation cohort from the MSKCC also confirmed the correlation between PTCH1 mutation and better prognosis (P = 0.022; HR, 0.290). Mechanically, diverse antitumor immune signatures were more highly enriched in PTCH1-mutated tumors than in PTCH1 wild-type tumors. Furthermore, PTCH1-mutated tumors had higher proportions of CD8 + T cells, activated NK cells, and M1 type macrophage infiltration, as well as elevated gene signatures of several steps in the cancer-immunity cycle. Notably, the PTCH1 mutation was correlated with tumor mutational burden (TMB), loss of heterozygosity score, and copy number variation burden. Our results show that the mutation of PTCH1 is a potential biomarker for predicting the response of CRC patients to immunotherapy.

Advances of biphenyl small-molecule inhibitors targeting PD-1/PD-L1 interaction in cancer immunotherapy.

Immunotherapy inhibiting the programmed death-1/programmed death ligand-1 (PD-1/PD-L1) interaction has emerged as one of the most attractive cancer treatment strategies. So far, the clinically used PD-1/PD-L1 inhibitors are monoclonal antibodies, but monoclonal antibodies have several limitations, such as poor pharmacokinetic properties, unchecked immune responses and high production cost. The development of small-molecule inhibitors targeting PD-1/PD-L1 interaction is showing great promise as a potential alternative or complementary therapeutic approach of monoclonal antibodies. In this article, the authors classify the reported biphenyl small-molecule inhibitors into symmetrical and asymmetrical types based on their structural features and further review their representative inhibitors and biological activities, as well as the binding models for providing insight into further exploration of more potent biphenyl small-molecule inhibitors targeting PD-1/PD-L1 interaction.

Intrapleural nano-immunotherapy promotes innate and adaptive immune responses to enhance anti-PD-L1 therapy for malignant pleural effusion.

Malignant pleural effusion (MPE) is indicative of terminal malignancy with a uniformly fatal prognosis. Often, two distinct compartments of tumour microenvironment, the effusion and disseminated pleural tumours, co-exist in the pleural cavity, presenting a major challenge for therapeutic interventions and drug delivery. Clinical evidence suggests that MPE comprises abundant tumour-associated myeloid cells with the tumour-promoting phenotype, impairing antitumour immunity. Here we developed a liposomal nanoparticle loaded with cyclic dinucleotide (LNP-CDN) for targeted activation of stimulators of interferon genes signalling in macrophages and dendritic cells and showed that, on intrapleural administration, they induce drastic changes in the transcriptional landscape in MPE, mitigating the immune cold MPE in both effusion and pleural tumours. Moreover, combination immunotherapy with blockade of programmed death ligand 1 potently reduced MPE volume and inhibited tumour growth not only in the pleural cavity but also in the lung parenchyma, conferring significantly prolonged survival of MPE-bearing mice. Furthermore, the LNP-CDN-induced immunological effects were also observed with clinical MPE samples, suggesting the potential of intrapleural LNP-CDN for clinical MPE immunotherapy.

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.

Platinum(IV) complexes as inhibitors of CD47-SIRPα axis for chemoimmunotherapy of cancer.

Phagocytosis of cancer cells by antigen presenting cells (APCs) is critical to activate the host's immune responses. However, the targeting ability of APCs to cancer cells is limited by the upregulation of transmembrane protein CD47 on the cancer cell surface. Blocking CD47 can affect the macrophage-mediated phagocytosis. Two platinum-based immunomodulators MUP and DMUP were synthesized to enhance the phagocytic activity of macrophages by blocking the CD47-SIRPα axis. These PtIV complexes not only showed high antiproliferative activity against a panel of human cancer cell lines, but also cooperated with human peripheral blood mononuclear cells (PBMCs) to suppress cancer cells. They acted as immune checkpoint inhibitors to modulate the immune responses of both cancer and immune cells. In particular, DMUP decreased the expression of CD47 in tumor tissues and promoted the polarization of macrophages from M2 to M1 phenotype in a mouse model of non-small cell lung cancer, thereby enhancing the anticancer effect. By interfering with DNA synthesis and stimulating immune system, DMUP takes the advantage of chemotherapy and immunotherapy to inhibit cancer cells. The dual efficacy of DMUP makes it a potential chemoimmunotherapeutic agent in cancer therapy.