Dual inhibitors of DNMT and HDAC remodels the immune microenvironment of colorectal cancer and enhances the efficacy of anti-PD-L1 therapy.

Colorectal cancer is the second most prevalent and deadly cancer worldwide. The emergence of immune checkpoint therapy has provided a revolutionary strategy for the treatment of solid tumors. However, less than 5% of colorectal cancer patients respond to immune checkpoint therapy. Thus, it is of great scientific significance to develop "potentiators" for immune checkpoint therapy. In this study, we found that knocking down different DNMT and HDAC isoforms could increase the expression of IFNs in colorectal cancer cells, which can enhance the effectiveness of immune checkpoint therapy. Therefore, the combined inhibition of DNMT and HDAC cloud synergistically enhance the effect of immunotherapy. We found that dual DNMT and HDAC inhibitors C02S could inhibit tumor growth in immunocompetent mice but not in immunocompromised nude mice, which indicates that C02S exerts its antitumor effects through the immune system. Mechanistically, C02S could increase the expression of ERVs, which generated the intracellular levels of dsRNA in tumor cells, and then promotes the expression of IFNs through the RIG-I/MDA5-MAVS signaling pathway. Moreover, C02S increased the immune infiltration of DCs and T cells in microenvironment, and enhanced the efficacy of anti-PD-L1 therapy in MC38 and CT26 mice model. These results confirmed that C02S can activate IFNs through the RIG-I/MDA5-MAVS signaling pathway, remodel the tumor immune microenvironment and enhance the efficacy of immune checkpoint therapy, which provides new evidence and solutions for the development of "potentiator" for colorectal cancer immunotherapy.

Discovery of novel nucleoside derivatives as selective lysine acetyltransferase p300 inhibitors for cancer therapy.

P300 and CBP are two closely related histone acetyltransferases that are important transcriptional coactivators of many cellular processes. Inhibition of the transcriptional regulator p300/CBP is a promising therapeutic approach in oncology. However, there are no reported single selective p300 or CBP inhibitors to date. In this study, we designed and optimized a series of lysine acetyltransferase p300 selective inhibitors bearing a nucleoside scaffold. Most compounds showed excellent inhibitory activity against p300 with IC50 ranging from 0.18 to 9.90 µM, except for J16, J29, J40, and J48. None of the compounds showed inhibitory activity against CBP (inhibition rate < 50 % at 10 µM). Then the cytotoxicity of the compounds against a series of cancer cells were evaluated. Compounds J31 and J32 showed excellent proliferation inhibitory activity on cancer cells T47D and H520 with desirable selectivity profile of p300 over CBP. These compounds could be promising lead compounds for the development of novel epigenetic inhibitors as antitumor agents.

Quinazoline-based hydroxamic acid derivatives as dual histone methylation and deacetylation inhibitors for potential anticancer agents.

Cancer is a common malignant disease with complex signaling networks, which means it is unmanageable to cancer therapy by using single classical targeted drug. Recently, dual- or multitarget drugs have emerged as a promising option for cancer therapies. Although many multifunctional compounds targeting HDAC have been validated, as far as we know, there is no molecule targeting GLP and HDAC synchronously. In the present work, we designed and synthesized a series of quinazoline-based hydroxamic acid derivatives as dual GLP and HDAC inhibitors. These hybrid compounds showed potent enzymatic inhibitory activities against GLP and HDAC1/6 with IC50 values in the nanomolar range of less than 190 nM. Furthermore, most of our compounds displayed significant broad spectrum cytotoxic activities apart from D3 and D8 against all the tested cancer cells with IC50 values less than 50 µM. D1, D6 and D7 showed more potent cytotoxic activities than D2, D4 and D5 in those cancer cells. Especially, compound D7 showed potent inhibitory potency activity against both GLP and HDAC1/6 with IC50 values of 1.3, 89, 13 nM. Besides, D7 exhibited the most potent antiproliferative activity against all the tested cancer cells. Further evaluations indicated that D7 could inhibit the methylation and deacetylation of H3K9 on protein level. Moreover, D7 could induce cancer cell apoptosis, G0/G1 cell cycle arrest, and partly block migration and invasion. All these thorough evaluations warranted D7 as a promising lead compound worth further optimization and development for cancer therapy.

Discovery of triazolo [1,5-a] pyridine derivatives as novel JAK1/2 inhibitors.

Small molecule JAK inhibitors have been demonstrated efficacy in rheumatoid arthritis, inflammatory bowel disease, and psoriasis with the approval of several drugs. Aiming to develop potent JAK1/2 inhibitors, two series of triazolo [1,5-a] pyridine derivatives were designed and synthesized by various strategies. The pharmacological results identified the optimized compounds J-4 and J-6, which exerted high potency against JAK1/2, and selectivity over JAK3 in enzyme assays. Furthermore, J-4 and J-6 effectively suppressed proliferation of JAK1/2 high-expression BaF3 cells accompanied with acceptable metabolic stability in liver microsomes. Therefore, J-4 and J-6 might serve as promising JAK1/2 inhibitors for further investigation.

Development of a versatile DNMT and HDAC inhibitor C02S modulating multiple cancer hallmarks for breast cancer therapy.

DNMT and HDAC are closely related to each other and involved in various human diseases especially cancer. These two enzymes have been widely recognized as antitumor targets for drug discovery. Besides, research has indicated that combination therapy consisting of DNMT and HDAC inhibitors exhibited therapeutic advantages. We have reported a DNMT and HDAC dual inhibitor 15a of which the DNMT enzymatic inhibitory potency needs to be improved. Herein we reported the development of a novel dual DNMT and HDAC inhibitor C02S which showed potent enzymatic inhibitory activities against DNMT1, DNMT3A, DNMT3B and HDAC1 with IC50 values of 2.05, 0.93, 1.32, and 4.16 µM, respectively. Further evaluations indicated that C02S could inhibit DNMT and HDAC at cellular levels, thereby inversing mutated methylation and acetylation and increasing expression of tumor suppressor proteins. Moreover, C02S regulated multiple biological processes including inducing apoptosis and G0/G1 cell cycle arrest, inhibiting angiogenesis, blocking migration and invasion, and finally suppressing tumor cells proliferation in vitro and tumor growth in vivo.

Design, synthesis and anticancer evaluation of acridine hydroxamic acid derivatives as dual Topo and HDAC inhibitors.

Multitarget inhibitors design has generated great interest in cancer treatment. Based on the synergistic effects of topoisomerase and histone deacetylase inhibitors, we designed and synthesized a new series of acridine hydroxamic acid derivatives as potential novel dual Topo and HDAC inhibitors. MTT assays indicated that all the hybrid compounds displayed good antiproliferative activities with IC50 values in low micromolar range, among which compound 8c displayed potent activity against U937 (IC50 = 0.90 µM). In addition, compound 8c also displayed the best HDAC inhibitory activity, which was several times more potent than HDAC inhibitor SAHA. Subsequent studies indicated that all the compounds displayed Topo II inhibition activity at 50 µM. Moreover, compound 8c could interact with DNA and induce U937 apoptosis. This study provides a suite of compounds for further exploration of dual Topo and HDAC inhibitors, and compound 8c can be a new dual Topo and HDAC inhibitory anticancer agent.

Synthesis and biological research of novel azaacridine derivatives as potent DNA-binding ligands and topoisomerase II inhibitors.

DNA and DNA-related enzymes are one of the most effective and common used intracellular anticancer targets in clinic and laboratory studies, however, most of DNA-targeting drugs suffered from toxic side effects. Development of new molecules with good antitumor activity and low side effects is important. Based on computer aided design and our previous studies, a series of novel azaacridine derivatives were synthesized as DNA and topoisomerases binding agents, among which compound 9 displayed the best antiproliferative activity with an IC50 value of 0.57µM against U937 cells, which was slightly better than m-AMSA. In addition, compound 9 displayed low cytotoxicity against human normal liver cells (QSG-7701), the IC50 of which was more than 3 times lower than m-AMSA. Later study indicated that all the compounds displayed topoisomerases II inhibition activity at 50µM. The representative compound 9 could bind with DNA and induce U937 apoptosis through the exogenous pathway.

Olaparib hydroxamic acid derivatives as dual PARP and HDAC inhibitors for cancer therapy.

Olaparib was the first PARP inhibitor approved by the FDA for patients with BRCA-mutated ovarian cancer. Recent studies have demonstrated enhanced anticancer effects of combination therapy consisting of olaparib and HDAC inhibitors. Herein, based on rational drug design strategy, hydroxamic acid derivatives of olaparib were constructed as dual PARP and HDAC inhibitors. These hybrid compounds showed potent inhibitory activities against PARP1/2 and HDAC1/6 with IC50 values in the nanomolar range. Furthermore, compound P1 exhibited broad-spectrum antiproliferative activities in selected human cancer cell lines. Specially, P1 showed more potent activity than olaparib and SAHA in cancer cells MDA-MB-231, HCC1937 and Raji, and 4.1-fold less cytotoxicity compared with SAHA to normal cells MCF-10A. Further mechanism study indicated that P1 could induce the cleavage of PARP and the hyperacetylation of histones, increase the expression of DNA damage biomarker γ-H2AX, decrease the level of BRCA1 and RAD51, and regulate tumor cell growth and apoptosis through modulating both mitochondrial- and death receptor-mediated pathways. Therefore, our study suggested that compounds targeting PARP and HDAC concurrently might be a practical approach for cancer therapy.

Bifunctional HDAC and DNMT inhibitor induces viral mimicry activates the innate immune response in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a unique breast cancer subtype characterized by a lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. Since TNBC lacks ER, PR, and HER2, there are currently no drugs that specifically target TNBC. Therefore, the development of new drugs or effective treatment strategies to target TNBC has become an urgent clinical need. Research has shown that the application of histone deacetylase (HDAC) inhibitors and DNA methyltransferase (DNMT) inhibitors leads to genomic and epigenomic instability. This, in turn, triggers the activation of pattern recognition receptors (PRRs) and subsequently activates downstream interferon (IFN) signalling pathways. In this study, the bifunctional HDAC and DNMT inhibitor J208 exhibited antitumour activity in TNBC cell lines. J208 effectively induced apoptosis and cell cycle arrest at the G0/G1 phase, inhibiting cell migration and invasion in TNBC. Moreover, this bifunctional inhibitor induced the expression of endogenous retroviruses (ERVs) and elicited a viral mimicry response, which increased the intracellular levels of double-stranded RNA (dsRNA) to activate the innate immune signalling pathway in TNBC. In summary, we demonstrated that the bifunctional inhibitor J208, which is designed to inhibit HDAC and DNMT, has potent anticancer effects, providing a new research basis for reactivating antitumour immunity by triggering innate immune signalling and offering a promising strategy for TNBC treatment.

Dual inhibitors of DNMT and HDAC induce viral mimicry to induce antitumour immunity in breast cancer.

The existing conventional treatments for breast cancer, including immune checkpoint blockade, exhibit limited effects in some cancers, particularly triple-negative breast cancer. Epigenetic alterations, specifically DNMT and HDAC alterations, are implicated in breast cancer pathogenesis. We demonstrated that DNMTs and HDACs are overexpressed and positively correlated in breast cancer. The combination of DNMT and HDAC inhibitors has shown synergistic antitumour effects, and our previously designed dual DNMT and HDAC inhibitor (termed DNMT/HDACi) 15a potently inhibits breast cancer cell proliferation, migration, and invasion and induces apoptosis in vitro and in vivo. Mechanistically, 15a induces a viral mimicry response by promoting the expression of endogenous retroviral elements in breast cancer cells, thus increasing the intracellular level of double-stranded RNA to activate the RIG-I-MAVS pathway. This in turn promotes the production of interferons and chemokines and augments the expression of interferon-stimulated genes and PD-L1. The combination of 15a and an anti-PD-L1 antibody had an additive effect in vivo. These findings indicate that this DNMT/HDACi has immunomodulatory functions and enhances the effectiveness of immune checkpoint blockade therapy. A novel dual DNMT and HDAC inhibitor induces viral mimicry, which induces the accumulation of dsRNA to activate tumoral IFN signalling and cytokine production to enhance the immune response in breast cancer.

Novel dual inhibitors of PARP and HDAC induce intratumoral STING-mediated antitumor immunity in triple-negative breast cancer.

PARP inhibitors and HDAC inhibitors have been approved for the clinical treatment of malignancies, but acquired resistance of or limited effects on solid tumors with a single agent remain as challenges. Bioinformatics analyses and a combination of experiments had demonstrated the synergistic effects of PARP and HDAC inhibitors in triple-negative breast cancer. A series of novel dual PARP and HDAC inhibitors were rationally designed and synthesized, and these molecules exhibited high enzyme inhibition activity with excellent antitumor effects in vitro and in vivo. Mechanistically, dual PARP and HDAC inhibitors induced BRCAness to restore synthetic lethality and promoted cytosolic DNA accumulation, which further activates the cGAS-STING pathway and produces proinflammatory chemokines through type I IFN-mediated JAK-STAT pathway. Moreover, these inhibitors promoted neoantigen generation, upregulated antigen presentation genes and PD-L1, and enhanced antitumor immunity when combined with immune checkpoint blockade therapy. These results indicated that novel dual PARP and HDAC inhibitors have antitumor immunomodulatory functions in triple-negative breast cancer. Novel dual PARP and HDAC inhibitors induce BRCAness to restore synthetic lethality, activating tumoral IFN signaling via the cGAS-STING pathway and inducing cytokine production, promoting neoantigen generation and presentation to enhance the immune response.

Design, synthesis and evaluation of novel ErbB/HDAC multitargeted inhibitors with selectivity in EGFRT790M mutant cell lines.

Acquired resistance leads to the failure of EGFR TKIs in NSCLC treatment. A novel series of hydroxamic acid-containing 4-aminoquinazoline derivatives as irreversible ErbB/HDAC multitargeted inhibitors for NSCLC therapy had been designed and synthesized, which displayed weak anti-proliferative activity in several EGFR wild-type cancer cell lines (NCI-H838, SK-BR-3, A549, A431) yet retained moderate activity to EGFRT790M resistance mutation harboring NCI-H1975 cells. The mechanistic studies revealed that the representative compound 11e was able to inhibit the phosphorylation of EGFR, up-regulate hyperacetylation of histone H3 and even reduce the expression of EGFR and Akt in NCI-H1975 cells. In further assays, compound 11e also showed moderate anti-proliferative activity in other EGFRT790M harboring tumor cell lines (NCI-H820, Ba/F3_EGFR_Del19-T790M-C797S) and low toxicities in normal cell lines (HL-7702, FHC). This selectivity of designed multitargeted compounds could serve as a potential strategy to circumvent multiple mechanisms of acquired resistance to EGFR-targeted therapy without severe toxicities and side effects resulting from broad inhibition.

Current discovery strategies for hepatocellular carcinoma therapeutics.

Introduction: The global incidence of hepatocellular carcinoma (HCC) is not expected to decline significantly over the next 30 years. And although the latest gene sequencing studies have established its genetic map, the potentially targetable drivers of HCC are, so far, difficult to identify. To date, only seven drugs have been approved by the FDA for unresectable HCC treatment; thus, effective therapeutic breakthroughs are still needed urgently.Areas covered: In this review, the authors discuss both genetic and epigenetic alterations in HCC and introduce the current progress with some of the representative molecular targeting inhibitors, listing some of the approved drugs for the targets of HCC. The structure-activity relationship of molecules (e.g. thalidomide, bortezomil) used for HCC is also discussed.Expert opinion: Effective therapeutic targets and effective drugs for HCC treatment are an urgent unmet need. Better understanding and characterization of genetic and epigenetic alterations, which are important to hepatocarcinogenesis, may help to understand the molecular pathogenesis of HCC, as well as provide novel therapeutic lead compounds for HCC treatment.

Phthalimide conjugations for the degradation of oncogenic PI3K.

PI3K/Akt/mTOR pathway is crucial for carcinogenesis and its inhibitors have made a great progress in cancer treatment. However, there is still a great developing space for PI3K inhibitors as the acquired drug resistance hindered their application in clinical. Proteolysis-targeting chimeras (PROTACs) with the potential to handle the challenges faced in drug development could be an alternative therapeutic strategy. Moreover, the past two years have witnessed remarkable advances in the development of phthalimide conjugation as a strategy for the degradation instead of inhibition of the targets, including BET family proteins, Sirtuin 2, CDK 9, Smad 3, and BCR-ABL proteins. Here, we designed and synthesized a series of potential small molecular PROTACs for the degradation of PI3K. Four compounds induced remarkable PI3K degradation and down-regulated the phosphorylation of Akt, S6K and GSK-3ß in liver cancer cells HepG2. Furthermore, the representative compound D proved to inhibit tumor cells proliferation by the induction of autophagy instead of apoptosis or cell cycle arrest.

Metabolic profiling on the effect of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in MCF-7 cells.

Polybrominated diphenyl ethers (PBDEs) are commonly used to prevent the development of fire in various factory products. Due to the adverse effects on human health and the bio-accumulation capacity, PBDEs are considered as one kind of persistent organic pollutants (POPs). BDE-47 is one of the most frequently detected PBDEs congeners in human samples. Although numerous studies have shown the close connection between BDE-47 and human health, few reports were related to breast carcinoma. In the present study, the toxicity mechanism of BDE-47 was investigated by using MCF-7 breast cancer cells. Metabolomics analysis was conducted by using ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS). Results showed that the toxicity to MCF-7 cells gradually increased when the concentration of BDE-47 exceeded 1 µM in the medium with 1% fetal bovine serum (FBS). It was found that pyrimidine metabolism, purine metabolism and pentose phosphate pathway (PPP) were the most influenced metabolic pathways, and the metabolites in the three metabolic pathways were significantly downregulated. Moreover, the increase of reactive oxygen species (ROS) was detected by using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method. The obtained results suggested that the BDE-47 induced oxidative stress by downregulating the NADPH generation in PPP. The pyrimidine metabolism and purine metabolism might be downregulated by the downregulation of mRNA transcripts. Therefore, BDE-47 could induce oxidative stress by inhibiting PPP and disorder the metabolism of the entire cell subsequently. This research provided evidence for investigating mechanism of the adverse effect of BDE-47 on human health.

Design, synthesis and biological evaluation of 4-amidobenzimidazole acridine derivatives as dual PARP and Topo inhibitors for cancer therapy.

PARP-1 could repair the DNA damages induced by Topo inhibitors, therefore inhibiting Topo and PARP-1 simultaneously might be able to overcome resistance and improve outcomes. In this study a series of 4-amidobenzimidazole acridines were designed and synthesized as dual Topo and PARP-1 inhibitors. Compound 11l displayed good inhibitory activities against Topo and PARP-1, as well as significantly inhibited cancer cells proliferation. Further mechanistic evaluations indicated that 11l treatment in MCF-7 cells induced accumulated DNA double-strand breaks, prompted remarkable apoptosis, and caused prominent G0/G1 cell cycle arrest. Moreover, 11l greatly suppressed tumor growth in mice, and displayed favorable metabolic properties in liver microsomes. Our study suggested that single agents inhibiting Topo and PARP concurrently might be an alternative for cancer therapy and 11l represented a potential lead compound for development of antitumor agents.

PARP inhibitors as antitumor agents: a patent update (2013-2015).

INTRODUCTION: PARP inhibitors have been extensively explored as antitumor agents and have shown potent efficacy both in vitro and in vivo. They can be used in monotherapy under the synthetic lethality concept or in combination with radiotherapy or chemotherapy, inducing a synergistic effect. Areas covered: This review covers relevant efforts in the development of PARP inhibitors with a particular focus on recently patented PARP inhibitors, combination therapy involving PARP inhibitors, tumor responsiveness to PARP inhibitors as detailed in reports made from 2013 - 2015, and PARP drugs in clinical trials and other novel inhibitors that emerged in 2013 - 2015. Expert opinion: Clinical studies and applications of PARP inhibitors as antitumor agents have gained considerable recognition in the last few years. In addition to FDA-approved olaparib, an increasing number of new inhibitors have been designed and synthesized, some of which are under preclinical or clinical evaluation. Novel inhibitors are still required, especially new scaffold compounds or drugs with improved properties, such as higher selectivity, higher potency and lower toxicity. The development of combination therapies involving PARP inhibitors and the exploration of biomarkers to predict outcomes with PARP inhibitors would expand the applications of these inhibitors, allowing more patients to benefit from this promising class of drugs in the future.

Design, synthesis and anticancer potential of NSC-319745 hydroxamic acid derivatives as DNMT and HDAC inhibitors.

DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) are important epigenetic targets during anticancer drug development. Recent study indicates that DNMT inhibitors and HDAC inhibitors display synergistic effects in certain cancers, therefore, development of molecules targeting both DNMT and HDAC is of therapeutic advantage against these cancers. Based on the structure of DNMT inhibitor NSC-319745 and the pharmacophore characteristics of HDAC inhibitors, a series of hydroxamic acid derivatives of NSC-319745 were designed and synthesized as DNMT and HDAC multifunctional inhibitors. Most compounds displayed potential DNMT inhibitory potency and potent HDAC inhibitory activity, especially compound 15a showed much better DNMT1 inhibitory potency than NSC-319745, and inhibited HDAC1, HDAC6 with IC50 values of 57, 17 nM, respectively. Furthermore, the synthesized compounds exhibited significant cytotoxicity against human cancer cells K562 and U937. Further mechanistic studies demonstrated that 15a treatment in U937 increased histones H3K9 and H4K8 acetylation, prompted P16 CpG islands demethylation and upregulated P16 expression, regulated apoptosis-related protein expression on the cellular level and induced remarkable U937 apoptosis. Moreover, genotoxicity of representative compounds was evaluated. In summary, our study provided a practical drug design strategy targeting multiple enzymes, and 15a represents a novel and promising lead compound for the development of novel epigenetic inhibitors as antitumor agents.