HVEM in acute lymphocytic leukemia facilitates tumour immune escape by inhibiting CD8+ T cell function.

PURPOSE: Leukaemia remains a major contributor to global mortality, representing a significant health risk for a substantial number of cancer patients. Despite notable advancements in the field, existing treatments frequently exhibit limited efficacy or recurrence. Here, we explored the potential of abolishing HVEM (herpes virus entry mediator, TNFRSF14) expression in tumours as an effective approach to treat acute lymphoblastic leukaemia (ALL) and prevent its recurrence. METHODS: The clinical correlations between HVEM and leukaemia were revealed by public data analysis. HVEM knockout (KO) murine T cell lymphoblastic leukaemia cell line EL4 were generated using CRISPR-Cas9 technology, and syngeneic subcutaneous tumour models were established to investigate the in vivo function of HVEM. Immunohistochemistry (IHC), RNA-seq and flow cytometry were used to analyse the tumour immune microenvironment (TIME) and tumour draining lymph nodes (dLNs). Immune functions were investigated by depletion of immune subsets in vivo and T cell functional assays in vitro. The HVEM mutant EL4 cell lines were constructed to investigate the functional domain responsible for immune escape. RESULTS: According to public databases, HVEM is highly expressed in patients with ALL and acute myeloid leukemia (AML) and is negatively correlated with patient prognosis. Genetic deletion of HVEM in EL4 cells markedly inhibited tumour progression and prolonged the survival of tumour-bearing mice. Our experiments proved that HVEM exerted its immunosuppressive effect by inhibiting antitumour function of CD8+ T cell through CRD1 domain both in vivo and in vitro. Additionally, we identified a combination therapy capable of completely eradicating ALL tumours, which induces immune memory toward tumour protection. CONCLUSIONS: Our study reveals the potential mechanisms by which HVEM facilitates ALL progression, and highlights HVEM as a promising target for clinical applications in relapsed ALL therapy.

FAP is a prognostic marker, but not a viable therapeutic target for clinical translation in HNSCC.

PURPOSE: PD-1 targeted immunotherapy has imparted a survival benefit to advanced head and neck squamous cell carcinoma (HNSCC), but less than 20% patients produce a durable response to this therapy. Here we aimed to investigate the potential biomarkers for predicting the clinical outcome and resistance to PD-1 targeted immunotherapy in HNSCC patients, and to examine the involvement of FAP+ cancer-associated fibroblasts (CAFs). METHODS: Bioinformatics methods were applied to analyze multiple datasets and explore the role of PD-1 and FAP in HNSCC. Immunohistochemistry was used to detect the expression of FAP protein. Fap gene knockout mice (Fap-/-) and L929 cells with different levels of Fap overexpression (L929-Fap-Low/High) were established to demonstrate the role of FAP+ CAFs in tumor development and immune checkpoint blockade (ICB) resistance. RESULTS: The expression level of PD-1 gene was positively correlated with better overall survival and therapeutic response to PD-1 blockade in HNSCC, but not all tumors with high expression of both PD-1 and PD-L1 were responsive. Moreover, FAP gene was overexpressed in pan-cancer tissues, and could serve as a prognostic biomarker for several cancers, including HNSCC. However, FAP protein was undetectable in mouse MTCQ1 tumors and barely expressed in human HNSCC tumors. Furthermore, FAP+ CAFs did not promote tumor growth or enhance the resistance to PD-1 inhibitor treatment. CONCLUSION: Although FAP+ CAFs have attracted increasing attention for their role in cancer, the feasibility and efficacy of FAP-targeting therapies for HNSCC remain doubtful.

mTORC1 signaling pathway integrates estrogen and growth factor to coordinate vaginal epithelial cells proliferation and differentiation.

The mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen. Estrogen acts as an activator of mTOR signaling but its role in vaginal epithelial homeostasis is unknown. We analyzed reproductive tract-specific Rptor or Rictor conditional knockout mice to reveal the role of mTOR signaling in estrogen-dependent vaginal epithelial cell proliferation and differentiation. Loss of Rptor but not Rictor in the vagina resulted in an aberrant proliferation of epithelial cells and failure of keratinized differentiation. As gene expression analysis indicated, several estrogen-mediated genes, including Pgr and Ereg (EGF-like growth factor) were not induced by estrogen in Rptor cKO mouse vagina. Moreover, supplementation of EREG could activate the proliferation and survival of vaginal epithelial cells through YAP1 in the absence of Rptor. Thus, mTORC1 signaling integrates estrogen and growth factor signaling to mediate vaginal epithelial cell proliferation and differentiation, providing new insights into vaginal atrophy treatment for post-menopausal women.

Design, synthesis, and biological evaluation of some novel 4-aminoquinazolines as Pan-PI3K inhibitors.

A series of 4-aminoquinazolines derivatives containing hydrophilic group were designed and identified as potent Pan-PI3K inhibitors in this study. The results of antiproliferative assays in vitro showed that this series of compounds had strong inhibition of tumor growth, especially compound 7b for MCF-7 cells but weak inhibition to normal cells. PI3K kinase assay showed that 7b had high activity for three PI3K isoforms with the IC50 values of picomole. The western blot assay indicated that 7b could decrease the phospho-Akt (S473) in a dose-dependent manner. Further experiments showed that 7b could induce apoptosis in MCF-7 cells. Four key hydrogen bonding interactions were found in the docking of 7b with PI3K kinase. All these results suggested that 7b is a potent PI3K inhibitor and could be considered as a potential candidate for the development of anticancer agents.

Design, synthesis and biological evaluation of novel series of 2H-benzo[b][1,4]oxazin-3(4H)-one and 2H-benzo[b][1,4]oxazine scaffold derivatives as PI3Kα inhibitors.

The abnormal activation of PI3K signaling pathway leads to the occurrence of various cancers. The PI3Kα is frequently mutated and overexpressed in many human cancers. Therefore, the PI3Kα was considered as a promising target in therapeutic treatment of cancer. In this study, two series of compounds containing 2H-benzo[b][1,4]oxazin-3(4H)-one and 2H-benzo[b][1,4]oxazine scaffold were synthesized and evaluated antiproliferative activities against three cancer cell lines, including HCT-116, MDA-MB-231 and SNU638. Compound 7f with the most potent antiproliferative activity was selected for further evaluation on normal cells and PI3K kinase. Studies indicated that compound 7f could decrease the phospho-Akt (T308) in a dose-dependent manner. Four key hydrogen bonding interactions were found in the docking of 7f with PI3K enzyme. All the results suggested that 7f was a potent PI3Kα inhibitor.

Novel 4-aminoquinazoline derivatives induce growth inhibition, cell cycle arrest and apoptosis via PI3Kα inhibition.

Phosphatidylinositol 3-kinase (PI3K) signaling pathway has diverse functions, including the regulation of cellular survival, proliferation, cell cycle, migration, angiogenesis and apoptosis. Among class I PI3Ks (PI3Kα, ß, γ, δ), the PIK3CA gene encoding PI3K p110α is frequently mutated and overexpressed in a large portion of human cancers. Therefore, the inhibition of PI3Kα has been considered as a promising target for the development of a therapeutic treatment of cancer. In this study, we designed and synthesized a series of 4-aminoquinazoline derivatives and evaluated their antiproliferative activities against six cancer cell lines, including HCT-116, SK-HEP-1, MDA-MB-231, SNU638, A549 and MCF-7. Compound 6b with the most potent antiproliferative activity and without obvious cytotoxicity to human normal cells was selected for further biological evaluation. PI3K kinase assay showed that 6b has selectivity for PI3Kα distinguished from other isoforms. The western blot assay and PI3K kinase assay indicated that 6b effectively inhibited cell proliferation via suppression of PI3Kα kinase activity with an IC50 of 13.6 nM and subsequently blocked PI3K/Akt pathway activation in HCT116 cells. In addition, 6b caused G1 cell cycle arrest owing to the inhibition of PI3K signaling and induced apoptosis via mitochondrial dependent apoptotic pathway. Our findings suggested that 6b has a therapeutic value as an anticancer agent via PI3Kα inhibition.

Design, synthesis and biological evaluation of novel 4-aminoquinazolines as dual target inhibitors of EGFR-PI3Kα.

The overexpression of EGFR correlates with rapidly progressive disease, resistance to chemotherapy and poor prognosis. In certain human cancers, PI3K works synergistically with EGFR to promote proliferation, survival, invasion and metastasis. Development of dual-target drugs against EGFR and PI3K has therapeutic advantage and was an attractive approach against tumors. In this work, based on the molecular docking and previous studies, a series of 4-aminoquinazolines derivatives containing 6-sulfonamide substituted pyridyl group were rationally designed and identified as potent EGFR and PI3K dual inhibitors. The cytotoxicity experiment results showed that this series of compounds could effectively inhibit cell growth. The kinase assay demonstrated that 6c and 6i had high inhibition for EGFR and selectivity for PI3Kα distinguished from other isoforms. Further experiments showed that 6c could induce cell cycle arrest in G1 phase and apoptosis in BT549 cells. The western blot assay indicated that 6c inhibited the proliferation of BT549 cell through EGFR and PI3Kα/Akt signaling pathway. Our study suggested that compound 6c was a potential dual inhibitors of EGFR and PI3Kα.

Design, synthesis, and biological evaluation of novel 3-substituted imidazo[1,2-a]pyridine and quinazolin-4(3H)-one derivatives as PI3Kα inhibitors.

Phosphatidylinositol 3-kinase (PI3K) is a pivotal regulator of intracellular signaling pathways and considered as a promising target in the development of a therapeutic treatment of cancer. Among the different PI3K subtypes, the PIK3CA gene encoding PI3K p110α is frequently mutated and overexpressed in majority of human cancers. Therefore, the inhibition of PI3Kα has been considered to be an efficient approach for the treatment of cancer. In this study, two series compounds containing hydrophilic group in imidazo[1,2-a]pyridine and quinazolin-4(3H)-one were synthesized and their antiproliferative activities against five cancer cell lines, including HCT-116, SK-HEP-1, MDA-MB-231, SNU638 and A549, were evaluated. Compound 1i with most potent antiproliferative activity was selected for further biological evaluation. PI3K kinase assay showed that 1i has selectivity for PI3Kα distinguished from other isoforms. The western blot assay indicated that 1i is more effective than HS-173, an imidazopyridine-based PI3Ka inhibitor, in reducing the levels of phospho-Akt. All these results suggested that 1i is a potent PI3Kα inhibitor and could be considered as a potential candidate for the development of anticancer agents.

Synthesis and Smo Activity of Some Novel Benzamide Derivatives.

Two series of benzamides compounds bearing piperidine groups were synthesized and the Gli-luc luciferase activity was screened by Gys-luc luciferase gene detection method. Compound 5q showed promising inhibition of hedgehog (Hh) signaling pathway. To further verify whether the Hh inhibitory activities of the target compounds are derived from their inhibition to the Smoothened (Smo) receptor, the compounds with good potency were evaluated in a fluorescence competitive displacement assays, the results showed the Smo inhibitory potency of these compounds correlated well with their Hh inhibition, which suggested that the observed Hh activity was driven by Smo inhibitors.

Versatile methods for synthesizing organic acid salts of quaternary berberine-type alkaloids as anti-ulcerative colitis agents.

Two versatile methods to synthesize kinds of organic acid salts of quaternary berberine-type alkaloids were investigated in order to determine which is more efficient to improve the liposolubility of the target compounds and to explore the efficacy of the target compounds as anti-ulcerative colitis (UC) agents. Overall evaluation according to the reaction results and yields of the final products indicated that the synthetic method using tertiary (±)-8-acylmethyldihydroberberine-type alkaloids as key intermediates is superior to that of using tertiary dihydroberberine-type alkaloids as intermediates. Ten target compounds were synthesized using quaternary berberine chloride and quaternary coptisine chloride as starting materials, respectively, and the anti-UC activity of some target compounds was evaluated in an in vitro x-box-binding protein 1 (XBP1) transcriptional activity assay using dual luciferase reporter detection. At 10 µM, the tested compounds were found to activate the transcription of XBP1 target at almost the same level as that of quaternary coptisine chloride. The synthesized target compounds were also found to share higher liposolubility than the inorganic acid salts of quaternary berberine-type alkaloid.

[A novel dipeptidyl peptidase IV inhibitors developed through scaffold hopping and drug splicing strategy].

Though all the marketed drugs of dipeptidyl peptidase IV inhibitors are structurally different, their inherent correlation is worthy of further investigation. Herein we rapidly discovered a novel DPP-IV inhibitor 8g (IC50 = 4.9 nmol.L-1) which exhibits as good activity and selectivity as the market drugs through scaffold hopping and drug splicing strategies based on alogliptin and linagliptin. This study demonstrated that the employment of classic medicinal chemistry strategy to the marketed drugs with specific target is an efficient approach to discover novel bioactive molecules.

Discovery of 2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide derivatives as new RORγ inhibitors using virtual screening, synthesis and biological evaluation.

Retinoic acid receptor-related orphan receptor γ (RORγ), a member of the nuclear hormone receptor superfamily, is a promising therapeutic target for treating Th17-mediated autoimmune diseases. We performed structure-based virtual screening targeting the RORγ ligand-binding domain. Among the tested compounds, s4 demonstrated RORγ antagonistic activities with micromolar IC50 values in both an AlphaScreen assay (20.27 µM) and a cell-based reporter gene assay (11.84 µM). Optimization of the s4 compound led to the identification of compounds 7j, 8c, 8k, and 8p, all of which displayed significantly enhanced RORγ inhibition with IC50 values of 40-140 nM. These results represent a promising starting point for developing potent small molecule RORγ inhibitors.

Discovery of novel urea derivatives as dual-target hypoglycemic agents that activate glucokinase and PPARγ.

Motivated by the discovery of a potential ligand that activates both glucokinase (GK) and perioxisome proliferator-activated receptor-γ (PPARγ), this work presents the rational design and synthesis of a series of novel urea derivatives as potent dual-target ligands of GK and PPARγ. The derivatives obtained, particularly compounds 14j, 14m, 15g, 15j, and 15s, showed relatively high enzyme activity and moderate blood glucose-lowering efficacy in normal ICR mice (GK activation fold >1.7, PPARγ activation percentage >38.8%, relative to rosiglitazone). The discovery of a dual-acting agent may provide an effective approach for treating type 2 diabetes mellitus.

Cellular mechanisms of a new pyrazinone compound that induces apoptosis in SKOV-3 cells.

We screened a small molecular library that was designed and independently synthesized in vitro and found a new drug (MY-03-01) that is active against ovarian cancer. We established that MY-03-01 effectively inhibited SKOV-3 cell survival in a dose-dependent manner, based on cell viability rates, and that it not only induced SKOV-3 apoptosis by itself, but also did so synergistically with paclitaxel. Secondly, when MY-03-01 was applied at 40 µM, its hemolytic activity was less than 10%, compared with the control, and there was almost no damage to normal cells at this concentration. In addition, we used DAPI staining and flow cytometry to show that MY- 03-01 could significantly induce apoptosis of SKOV-3 cells. Finally, we found that MY-03-01 likely induced SKOV-3 apoptosis by activating caspase3 and caspase9 through the mitochondrial pathway.

[Design, synthesis and biological evaluation of novel diaryl ethers bearing a pyrimidine motif as human Pin1 inhibitors].

Pin1 (peptidyl-prolyl cis-trans isomerase NIMA-interacting 1) belongs to peptidyl-prolyl cis-trans isomerase (PPIase) and is a novel promising anticancer target. Based on the lead structure of benzophenone, a series of novel diarylether derivatives containing a pyrimidine ring were designed and synthesized. The inhibitory activities on Pin1 of compounds 5a-5d and 6a-6i were evaluated by a protease-coupled enzyme assay. Of all the evaluated compounds, 6 compounds displayed inhibitory activities. Molecular docking was performed using FlexX algorithm to explore the binding mode of the active molecules.

[Salicylic acid derivatives as simplified and novel GK small molecule activators].

Glucokinase (GK) is a new target for the treatment of type II diabetes mellitus (T2DM). In order to find a structure-simplified small molecule GK activator, 19 salicylic acid derivatives were designed and synthesized based on new lead compound (1). Experimental results showed that the potency of compound 8h is superior to control RO-28-0450 in GK activation.

Design, synthesis and biological evaluation of N-sulfonyl homoserine lactone derivatives as inhibitors of quorum sensing in Chromobacterium violaceum.

A novel series of N-sulfonyl homoserine lactone derivatives 5a-l has been designed, synthesized and evaluated for quorum sensing inhibitory activities towards violacein production. Of the compounds synthesized, compound 5h was found to possess an excellent level of enantiopurity (99.2% e.e.). The results indicated that compounds bearing an ortho substituent on their phenyl ring exhibited excellent levels of inhibitory activity against violacein production. Compounds 5h and 5k in particular, with IC50 values of 1.64 and 1.66 µM, respectively, were identified as promising lead compounds for further structural modification.

Synthesis and cytotoxic activity of some novel N-pyridinyl-2-(6-phenylimidazo[2,1-b]thiazol-3-yl)acetamide derivatives.

A series of novel compounds bearing imidazo[2,1-b]thiazole scaffolds were designed and synthesized based on the optimization of the virtual screening hit compound N-(6-morpholinopyridin-3-yl)-2-(6-phenylimidazo[2,1-b]thiazol-3-yl)acetamide (5a), and tested for their cytotoxicity against human cancer cell lines, including HepG2 and MDA-MB-231. The results indicated that the compound 2-(6-(4-chlorophenyl)imidazo[2,1-b]thiazol-3-yl)-N-(6-(4-(4-methoxybenzyl)piperazin-1-yl)pyridin-3-yl)acetamide (5l), with slightly higher inhibition on VEGFR2 than 5a (5.72% and 3.76% inhibitory rate at 20 µM, respectively), was a potential inhibitor against MDA-MB-231 (IC(50) = 1.4 µM) compared with sorafenib (IC(50) = 5.2 µM), and showed more selectivity against MDA-MB-231 than HepG2 cell line (IC(50) = 22.6 µM).

Synthesis and biological evaluation of novel human Pin1 inhibitors with benzophenone skeleton.

A series of novel benzophenone derivatives were prepared and their inhibitory activities were evaluated on hPin1. Of all the synthesized compounds, the most active compound displayed inhibitory activities with an IC(50) value of 5.99 µmol/L. Preliminary structure-activity relationships were analyzed in details and the binding mode of the titled compounds was predicted using FlexX algorithm. The results of this research will shed light on further design and optimization of novel small molecule Pin1 inhibitors.

[Design, synthesis and antitumor activity of sorafenib analogues containing 2-picolinylhydrazide moiety].

A novel series of sorafenib analogs containing 2-picolinyl hydrazide moiety were designed and synthesized. In vitro, most of synthesized compounds have antiproliferation activity on MDA-MB-231, ACHN, HepG2, Mia-PaCa-2 and SW1990 cell lines tested by MTT assay. It is worth noting that the antitumor activities of compounds 2c, 2d and 2f are more potent than that of sorafenib on pancreatic cancer cells Mia-PaCa-2 and SW1990, and the activities of compounds 3f and 3g are 2-3 times than that of sorafenib on human hepatocellular carcinoma HepG2 cell line.