G-quadruplex DNA structure is a positive regulator of MYC transcription.

DNA structure can regulate genome function. Four-stranded DNA G-quadruplex (G4) structures have been implicated in transcriptional regulation; however, previous studies have not directly addressed the role of an individual G4 within its endogenous cellular context. Using CRISPR to genetically abrogate endogenous G4 structure folding, we directly interrogate the G4 found within the upstream regulatory region of the critical human MYC oncogene. G4 loss leads to suppression of MYC transcription from the P1 promoter that is mediated by the deposition of a de novo nucleosome alongside alterations in RNA polymerase recruitment. We also show that replacement of the endogenous MYC G4 with a different G4 structure from the KRAS oncogene restores G4 folding and MYC transcription. Moreover, we demonstrate that the MYC G4 structure itself, rather than its sequence, recruits transcription factors and histone modifiers. Overall, our work establishes that G4 structures are important features of transcriptional regulation that coordinate recruitment of key chromatin proteins and the transcriptional machinery through interactions with DNA secondary structure, rather than primary sequence.

Unveiling the role of G-quadruplex structure in promoter region: Regulation of ABCA1 expression in macrophages possibly via NONO protein recruitment.

ABCA1 has been found to be critical for cholesterol efflux in macrophages. Understanding the mechanism regulating ABCA1 expression is important for the prevention and treatment of atherosclerosis. In the present study, a G-quadruplex (G4) structure was identified in the ABCA1 promoter region. This G4 was shown to be essential for ABCA1 transcription. Stabilizing the G4 by ligands surprisingly upregulated ABCA1 expression in macrophages. Knocking out the G4 remarkably reduced ABCA1 expression, and abolished the increase of ABCA1 expression induced by the G4 ligand. By pull-down assays, the protein NONO was identified as an ABCA1 G4 binder. Overexpression or repression of NONO significantly induced upregulation and downregulation of ABCA1 expression, respectively. ChIP and EMSA experiments showed that the G4 ligand promoted the binding between the ABCA1 G4 and NONO, which led to more recruitment of NONO to the promoter region and enhanced ABCA1 transcription. Finally, the G4 ligand was shown to significantly reduce the accumulation of cholesterol in macrophages. This study showed a new insight into the regulation of gene expression by G4, and provided a new molecular mechanism regulating ABCA1 expression in macrophages. Furthermore, the study showed a possible novel application of the G4 ligand: preventing and treating atherosclerosis.

A Novel PDK1/MEK Dual Inhibitor Induces Cytoprotective Autophagy via the PDK1/Akt Signaling Pathway in Non-Small Cell Lung Cancer.

In a preliminary study, we synthesized a series of new PDK1/MEK dual inhibitors. Antitumor activity screening showed that Compound YZT exerts a strong inhibitory action in A549 cells. However, the specific mechanism of YZT against non-small cell lung cancer (NSCLC) is largely unknown. This work confirmed the anti-proliferation and pro-apoptosis effects of YZT in NSCLC cells. Furthermore, YZT promotes autophagy and provokes complete autophagic flux in NSCLC cells. Notably, compared with YZT alone, the combination of YZT with the autophagy inhibitor chloroquine (CQ) or 3-methyladenine (3-MA) markedly strengthened the anti-proliferative and pro-apoptotic actions, suggesting that YZT-induced autophagy is cytoprotective. We further found that YZT-induced autophagy may exert a cytoprotective function by preserving the integrity of mitochondria and decreasing mitochondrial apoptosis. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that PDK1 is an upstream protein of the Akt/mTOR axis and western blotting verified that YZT induces autophagy by the PDK1/Akt/mTOR signaling axis. Finally, YZT plus CQ significantly enhanced the anticancer activities compared to YZT alone in an animal study and immunohistochemistry showed that the level of LC3 was increased by YZT, which is in line with the in vitro results. In short, our study provides reliable experimental basis for developing Compound YZT as a new chemotherapeutic drug candidate and suggests that combined administration of YZT with CQ is a potential therapy against NSCLC.

Chem-map profiles drug binding to chromatin in cells.

Characterizing drug-target engagement is essential to understand how small molecules influence cellular functions. Here we present Chem-map for in situ mapping of small molecules that interact with DNA or chromatin-associated proteins, utilizing small-molecule-directed transposase Tn5 tagmentation. We demonstrate Chem-map for three distinct drug-binding modalities as follows: molecules that target a chromatin protein, a DNA secondary structure or that intercalate in DNA. We map the BET bromodomain protein-binding inhibitor JQ1 and provide interaction maps for DNA G-quadruplex structure-binding molecules PDS and PhenDC3. Moreover, we determine the binding sites of the widely used anticancer drug doxorubicin in human leukemia cells; using the Chem-map of doxorubicin in cells exposed to the histone deacetylase inhibitor tucidinostat reveals the potential clinical advantages of this combination therapy. In situ mapping with Chem-map of small-molecule interactions with DNA and chromatin proteins provides insights that will enhance understanding of genome and chromatin function and therapeutic interventions.

A novel dual MEK/PDK1 inhibitor 9za retards the cell cycle at G0/G1 phase and induces mitochondrial apoptosis in non-small cell lung cancer cells.

BACKGROUND: A novel dual MEK/PDK1 inhibitor named 9za has been synthesized by our research team. Preliminary study showed that 9za possessed potent cytotoxicity and proapoptosis in non-small cell lung cancer (NSCLC) cells. Nevertheless, the precise underlying mechanism is vague. METHODS: In this work, we adopted the MTT assay, the Cell Cycle Detection Kit, and the JC-1 staining assay to detect the cell viability, the cell cycle distribution and the mitochondrial membrane potential (MMP), respectively. Cell apoptosis was measured by the morphology observation under a light microscope, Annexin V-FITC/propidium iodide (PI) apoptosis detection and the colorimetric TUNEL assay. Western blot was used to monitor the cell cycle-, apoptosis-related proteins and relevant proteins involved in the signaling pathways. RESULTS: The MTT assay demonstrated that 9za sharply decreased the viability of NSCLC cells. Cell cycle analysis revealed that low concentrations of 9za arrested the cell cycle at the G0/G1 phase , which was further confirmed by the decreased levels of Cyclin D1, cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6). Additionally, morphological observations, Annexin V-FITC/propidium iodide (PI) apoptosis analysis and TUNEL assays indicated that high concentrations of 9za induced cell apoptosis. Furthermore, the JC-1 staining assay revealed that the mitochondrial membrane potential was downregulated following 9za exposure. Western blot also showed that 9za markedly decreased the expression levels of total Bcl-2, Cytochrome C in the mitochondria and BCL2 associated X (BAX) in the cytoplasm. However, the levels of BAX in the mitochondria, Cytochrome C in the cytoplasm, active caspase-9, active caspase-3 and cleaved-PARP showed the opposite changes. Moreover, the dose-dependent decreased phosphorylation levels of PDK1, protein kinase B (Akt), MEK and extracellular signal regulated kinase 1/2 (ERK1/2) after 9za treatment verified that 9za was indeed a dual MEK/PDK1 inhibitor, as we expected. Compared with a single MEK inhibitor PD0325901 or a single PDK1 inhibitor BX517, the dual MEK/PDK1 inhibitor 9za could strengthen the cytotoxic and proapoptotic effect, indicating that the double blocking of the MEK and PDK1 signaling pathways plays stronger cell growth inhibition and apoptosis induction roles than the single blocking of the MEK or PDK1 signaling pathway in NSCLC cells. Our work elucidated the molecular mechanisms for 9za as a novel drug candidate against NSCLC.

Current advances in idiopathic pulmonary fibrosis: the pathogenesis, therapeutic strategies and candidate molecules.

Idiopathic pulmonary fibrosis (IPF) is a type of chronic, progressive lung disease with unknown cause, which is characterized by increasing dyspnea and destruction of lung function with a high mortality rate. Evolving evidence demonstrated that the pathogenesis of IPF involved multiple signaling pathways such as inflammation, oxidative stress and fibrosis. However, drug discovery to prevent or revert IPF has been insufficient to cope with the development. Drug discovery targeting multiple links should be considered. In this review, we will brief the pathogenesis of IPF and discuss several small chemical entities toward the pathogenesis for IPF studied in animal models and clinical trials. The field of novel anti-IPF agents and the future directions for the prevention and treatment of IPF are detailed thoroughly discussed.

Binding pocket-based design, synthesis and biological evaluation of novel selective BRD4-BD1 inhibitors.

Bromodomain-containing protein 4 (BRD4), consisting of two tandem bromodomains (BD1 and BD2), is key epigenetic regulator in fibrosis and cancer, which has been reported that BD1 and BD2 have distinct roles in post-translational modification. But there are few selective inhibitors toward those two domains. Herein, this study designed and synthesized a series of novel selective BRD4-BD1 inhibitors, using computer-aided drug design (CADD) approach focused on exploring the difference of the binding pockets of BD1 and BD2, and finding the His437 a crucial way to achieve BRD4-BD1 selectivity. Our results revealed that the compound 3u is a potent selective BRD4-BD1 inhibitor with IC50 values of 0.56 µM for BD1 but >100 µM for BD2. The compound exhibited a broad spectrum of anti-proliferative activity against several human cancer and fibroblastic cell lines, which might be related to its capability of reducing the expression of c-Myc and collagen I. Furthermore, it could induce apoptosis in A375 cells. To the contrary, the selective BD2 inhibitor, RVX-208, did not indicate any of these activities. Our findings highlight that the function of BRD4-BD1 might be predominant in fibrosis and cancer. And it is rational to further develop novel selective BRD4-BD1 inhibitors.

Dual inhibitors of RAF-MEK-ERK and PI3K-PDK1-AKT pathways: Design, synthesis and preliminary anticancer activity studies of 3-substituted-5-(phenylamino) indolone derivatives.

The dysfunction and mutual compensatory activation of RAF-MEK-ERK and PI3K-PDK1-AKT pathways have been demonstrated as the hallmarks in several primary and recurrent cancers. The strategy of concurrent blocking of these two pathways shows clinical merits on effective cancer therapy, such as combinatory treatments and dual-pathway inhibitors. Herein, we report a novel prototype of dual-pathway inhibitors by means of merging the core structural scaffolds of a MEK1 inhibitor and a PDK1 inhibitor. A library of 43 compounds that categorized into three series (Series I-III) was synthesized and tested for antitumor activity in lung cancer cells. The results from structure-activity relationship (SAR) analysis showed the following order of antitumor activity that 3-hydroxy-5-(phenylamino) indolone (Series III) > 3-alkenyl-5-(phenylamino) indolone (Series I) > 3-alkyl-5-(phenylamino) indolone (Series II). A lead compound 9za in Series III showed most potent antitumor activity with IC50 value of 1.8 ±â€¯0.8 µM in A549 cells. Moreover, antitumor mechanism study demonstrated that 9za exerted significant apoptotic effect, and cellular signal pathway analysis revealed the potent blockage of phosphorylation levels of ERK and AKT in RAF-MEK-ERK and PI3K-PDK1-AKT pathways, respectively. The results reported here provide robust experimental basis for the discovery and optimization of dual pathway agents for anti-lung cancer therapy.

Recent Progress of Targeted G-Quadruplex-Preferred Ligands Toward Cancer Therapy.

A G-quadruplex (G4) is a well-known nucleic acid secondary structure comprising guanine-rich sequences, and has profound implications for various pharmacological and biological events, including cancers. Therefore, ligands interacting with G4s have attracted great attention as potential anticancer therapies or in molecular probe applications. To date, a large variety of DNA/RNA G4 ligands have been developed by a number of laboratories. As protein-targeting drugs face similar situations, G-quadruplex-interacting drugs displayed low selectivity to the targeted G-quadruplex structure. This low selectivity could cause unexpected effects that are usually reasons to halt the drug development process. In this review, we address the recent research on synthetic G4 DNA-interacting ligands that allow targeting of selected G4s as an approach toward the discovery of highly effective anticancer drugs.

Pip-HoGu: An Artificial Assembly with Cooperative DNA Recognition Capable of Mimicking Transcription Factor Pairs.

Cooperation between pairs of transcription factors (TFs) has been widely demonstrated to play a pivotal role in the spatiotemporal regulation of gene expression, but blocking cooperative TF pair-DNA interactions synergistically has been challenging. To achieve this, we designed programmable DNA binder pyrrole-imidazole polyamides conjugated to host-guest assemblies (Pip-HoGu) to mimic the cooperation between natural TF pairs. By incorporating cyclodextrin (Cyd)-adamantane (Ada), we synthesized Ada1 (PIP1-Ada) and Cyd1 (PIP2-Cyd), which were evaluated using Tm, EMSA, competitive, and SPR assays and molecular dynamics studies. The results consistently demonstrated that Pip-HoGu system formed stable noncovalent cooperative complexes, thereby meeting key criteria for mimicking a TF pair. The system also had a longer recognition sequence (two-PIP binding length plus gap distance), favorable sequence selectivity, higher binding affinity, and in particular, a flexible gap distance (0-5 bp). For example, Ada1-Cyd1 showed thermal stability of 7.2 °C and a minimum free energy of interaction of -2.32 kcal·mol-1 with a targeting length of 14 bp. Furthermore, cell-based evaluation validated the capability of Pip-HoGu to exhibit potent cooperative inhibitory effects on gene expression under physiological conditions by disrupting TF pair-DNA function. In conclusion, the modular design of Pip-HoGu defines a general framework for mimicking naturally occurring cooperative TF pair-DNA interactions that offers a promising strategy for applications in the precise manipulation of cell fate.

Antiproliferative and apoptotic activities of sequence-specific histone acetyltransferase inhibitors.

In parallel to monomeric epigenetic regulators, sequence-specific epigenetic regulators represent versatile synthetic dual-target ligands that achieve regulatory control over multi-gene networks. Development of DNA-binding domain (DBD)-HDAC inhibitors and DBD-HAT activators, which result in increased histone acetylation, has become one promising research field. However, there is no report regarding the gene regulatory pattern by sequence-specific epigenetic repressor. We report here for the first time, the synthesis of DBD-HAT inhibitors and demonstrate that these conjugates could retain their dual-target activity using predicted working model of thermal stability assay and in vitro HAT activity assay. Evaluation of antiproliferative activity in cancer cells showed that 2 (with a medium linker length of 13-atom) exhibited the highest antiproliferative activity in p53 wild-type cancer cell lines (IC50 of 1.8-2.6 µM in A549 and MV4-11 cells) and not in p53 mutant cancer cell lines. A mechanistic investigation using microarray analysis and an apoptotic assay showed that the antiproliferative effect of 2 occurred via the up-regulation of p53 target genes, and the subsequent initiation of p53-dependent apoptosis. Our research on sequence-specific dual-target epigenetic repressor offers us an alternative way to modulate HAT-governed therapeutically important genes and contributes to offer a fresh insight into antitumor therapeutics.

The RAF-MEK-ERK pathway: targeting ERK to overcome obstacles to effective cancer therapy.

AIM: Currently, dozens of BRAF inhibitors and MEK inhibitors targeting RAF-MEK-ERK pathway have been introduced into clinical trials for cancer therapy. However, after 6-8 months of initial response, acquired drug resistance among the majority of those treated patients sharply diminished their clinical efficacy. DISCUSSION: Important mechanisms responsible for acquired resistance of BRAF inhibitors and MEK inhibitors have been elucidated. Continually, ERK1/2 locates in the critical position and features unique characteristics, such as activating hundreds of substrates, participating in feedback regulation, being catalyzed by MEK specifically and no acquired resistant mutation. CONCLUSION: Taking in account the inspiring outcomes of ERK inhibitors in preclinical research, ERK1/2 might be the optimal target to overcome acquired drug resistance in RAF-MEK-ERK pathway.