A series of novel azole-diphenylpyrimidine derivatives (AzDPPYs) were synthesized and biologically evaluated as potent EGFRT790M inhibitors. Among these analogues, the most active inhibitor 6e not only displayed high activity against EGFRT790M/L858R kinase (IC50=3.3nM), but also was able to repress the replication of H1975 cells harboring EGFRT790M mutation at a concentration of 0.118μmol/L. In contrast to the lead compound rociletinib, 6e slightly reduces the key EGFRT790M-minduced drug resistance. Significantly, inhibitor 6e demonstrates high selectivity (SI=299.3) for T790M-containing EGFR mutants over wild type EGFR, hinting that it will cause less side effects.

MicroRNAs (miRNAs) regulate apoptosis, yet their role in regulated necrosis remains unknown. miR-21 is overexpressed in nearly all human cancer types and its role as an oncogene is suggested to largely depend on its anti-apoptotic action. Here we show that miR-21 is overexpressed in a murine model of acute pancreatitis, a pathologic condition involving RIP3-dependent regulated necrosis (necroptosis). Therefore, we investigate the role of miR-21 in acute pancreatitis injury and necroptosis. miR-21 deficiency protects against caerulein- or L-arginine-induced acute pancreatitis in mice. miR-21 inhibition using locked-nucleic-acid-modified oligonucleotide effectively reduces pancreatitis severity. miR-21 deletion is also protective in tumour necrosis factor-induced systemic inflammatory response syndrome. These data suggest that miRNAs are critical participants in necroptosis and miR-21 enhances cellular necrosis by negatively regulating tumour suppressor genes associated with the death-receptor-mediated intrinsic apoptosis pathway, and could be a therapeutic target for preventing pathologic necrosis.

Phosphatidylinositol 4,5-bisphosphate (PIP2) directly interacts with the small-conductance Ca2+-activated K+ 2-a (SK2-a) channel/calmodulin complex, serving as a critical element in the regulation of channel activity. We report that changes of protein conformation in close proximity to the PIP2 binding site induced by a small-molecule SK channel modulator, NS309, can effectively enhance the interaction between the protein and PIP2 to potentiate channel activity. This novel modulation of PIP2 sensitivity by small-molecule drugs is likely not to be limited in its application to SK channels, representing an intriguing strategy to develop drugs controlling the activity of the large number of PIP2-dependent proteins.

Multidrug resistance(MDR) is a major obstacle to efficiency of breast cancer chemotherapy. We investigated whether combination of metformin and 2-deoxyglucose reverses MDR of MCF-7/Dox cells and tried to elucidate the possible mechanisms. The combination of metformin and 2-deoxyglucose selectively enhanced cytotoxicity of doxorubicin against MCF-7/Dox cells. Combination of the two drugs resumed p53 function via inhibiting overexpression of murine doubleminute 2(MDM2) and murine doubleminute 4(MDM4) leading to G2/M arrest and apoptosis in MCF-7/Dox cells. Combination of the two drugs had no effect on P-glycoprotein mRNA expression and P-glycoprotein ATPase activity but increased doxorubicin accumulation in MCF-7/Dox cells. The increased doxorubicin accumulation maybe associate with metabolic stress. Combination of metformin and 2-deoxyglucose initiated a strong metabolic stress in MCF-7/Dox cells via inhibiting glucose uptake, lactate, fatty acid, ATP production and protein kinase B(AKT)/ mammalian target of rapamycin(mTOR) pathway. Taken together, combination of metformin and 2-deoxyglucose reverses MDR of MCF-7/Dox cells by recovering p53 function and increasing doxorubicin accumulation. Furthermore, doxorubicin selectively increases MCF-7/Dox apoptosis via aggravating metabolic stress induced by metformin plus 2-deoxyglucose. The mutually reinforcing effect made the combination of metformin and 2DG had a better effect on reversing MDR.

Arsenic is a well-known of human carcinogen and miR-301a is an oncogenic microRNA, which links to oncogenesis, however, little is understood about its contribution to arsenic-induced cellular transformation and tumorigenesis. Here, we investigated the role of miR-301a during arsenic-induced cellular transformation and tumor formation. miR-301a was found to be upregulated during arsenic-induced BEAS-2B transformation and the overexpression of miR-301a was dependent on IL-6/STAT3 signaling. Inhibition of miR-301a leads to reduction of cell proliferation, colony formation and cell migration. By using dual luciferase assay, SMAD4 was confirmed to be a direct target of miR-301a in BEAS-2B cells and upregulation of SMAD4 is involved the restraining cell growth and migration. In addition, reducing of miR-301a expression enhances doxorubicin-induced cellular apoptosis of transformed BEAS-2B through up-regulating SMAD4. Furthermore, we demonstrated that downregulation of miR-301a in BEAS-2B attenuates tumor growth in the xenograft model by targeting SMAD4. Of note, the level of miR-301a expression correlated inversely with SMAD4 expression in clinical specimens of human lung cancer. Our findings ascertain that miR-301a is an oncogenic miRNA, which targets SMAD4 to establish an essential mechanism for arsenic-induced carcinogenesis, IL-6/STAT3/miR-301a/SMAD4 signaling pathways.

A new family of diphenylpyrimidine derivatives bearing an amino acid substituent were identified as potent BTK inhibitors. Among them, compound 7b, which features an l-proline substituent, was identified as the strongest BTK inhibitor, with an IC50 of 8.7 nM. Compound 7b also displayed similar activity against B-cell lymphoma cell lines as ibrutinib. Moreover, 7b exhibited low cytotoxic activity against normal PBMC cells. In addition, the acridine orange/ethidium bromide (AO/EB) staining assay, Western blot analysis and flow cytometry analysis also showed its effectiveness in interfering with B-cell lymphoma cell growth. The molecular simulation performance showed that 7b forms additional strong hydrogen bonds with the BTK protein. All these findings provided new clues about the pyrimidine scaffold as an effective BTK inhibitor for the treatment of B-cell lymphoma.

The slow delayed rectifier (I(KS)) channel is composed of KCNQ1 (pore-forming) and KCNE1 (auxiliary) subunits, and functions as a repolarization reserve in the human heart. Design of I(KS)-targeting anti-arrhythmic drugs requires detailed three-dimensional structures of the KCNQ1/KCNE1 complex, a task made possible by Kv channel crystal structures (templates for KCNQ1 homology-modeling) and KCNE1 NMR structures. Our goal was to build KCNQ1/KCNE1 models and extract mechanistic information about their interactions by molecular-dynamics simulations in an explicit lipid/solvent environment. We validated our models by confirming two sets of model-generated predictions that were independent from the spatial restraints used in model-building. Detailed analysis of the molecular-dynamics trajectories revealed previously unrecognized KCNQ1/KCNE1 interactions, whose relevance in I(KS) channel function was confirmed by voltage-clamp experiments. Our models and analyses suggest three mechanisms by which KCNE1 slows KCNQ1 activation: by promoting S6 bending at the Pro hinge that closes the activation gate; by promoting a downward movement of gating charge on S4; and by establishing a network of electrostatic interactions with KCNQ1 on the extracellular surface that stabilizes the channel in a pre-open activated state. Our data also suggest how KCNE1 may affect the KCNQ1 pore conductance.

A new series of diphenylpyrimidine derivatives (DPPYs) bearing various aniline side chains at the C-2 position of pyrimidine core were synthesized as potent BTK inhibitors. Most of these inhibitors displayed improved activity against B leukemia cell lines compared with lead compound spebrutinib. Subsequent studies showed that the peculiar inhibitor 7j, with IC50 values of 10.5 μM against Ramos cells and 19.1 μM against Raji cells, also displayed slightly higher inhibitory ability than the novel agent ibrutinib. Moreover, compound 7j is not sensitive to normal cells PBMC, indicating low cell cytotoxicity. In addition, flow cytometry analysis indicated that 7j significantly induced the apoptosis of Ramos cells, and arrested the cell cycle at the G0/G1 phase. These explorations provided new clues to discover pyrimidine scaffold as more effective BTK inhibitors.

Previous studies have indicated that large intergenic non-coding RNA regulator of reprogramming (lincRNA-ROR) plays an important role in regulating tumor carcinogenesis and metastasis; however, whether circulating lincRNA-ROR could function as a potential biomarker for breast cancer (BC) diagnosis and monitoring is unknown. This study was conducted to investigate circulating lincRNA-ROR in plasma as a potential biomarker for BC diagnosis and monitoring.

Resveratrol, a natural phytoalexin, exhibits a remarkable range of biological activities, such as anticancer, cardioprotective, neuroprotective and antioxidant properties. However, the therapeutic application of resveratrol was encumbered for its low bioavailability. Therefore, many researchers focused on designing and synthesizing the derivatives of resveratrol to enhance the bioavailability and the pharmacological activity of resveratrol. During the past decades, a large number of natural and synthetic resveratrol derivatives were extensively studied, and the methoxylated, hydroxylated and halogenated derivatives of resveratrol received particular more attention for their beneficial bioactivity. So, in this review, we will summarize the chemical structure and the therapeutic versatility of resveratrol derivatives, and thus provide the related structure activity relationship reference for their practical applications.

With the aim to overcome the drug resistance induced by the EGFR T790M mutation (EGFRT790M), herein, a family of diphenylpyrimidine derivatives (Sty-DPPYs) bearing a C-2 (E)-4-(styryl)aniline functionality were designed and synthesized as potential EGFRT790M inhibitors. Among them, the compound 10e displayed strong potency against the EGFRT790M enzyme, with the IC50 of 11.0nM. Compound 10e also showed a higher SI value (SI=49.0) than rociletinib (SI=21.4), indicating its less side effect. In addition, compound 10e could effectively inhibit the proliferation of H1975 cells harboring the EGFRT790M mutation, within the concentration of 2.91μM. Significantly, compound 10e has low toxicity against the normal HBE cell (IC50=22.48μM). This work provided new insights into the discovery of potent and selective inhibitor against EGFRT790M over wild-type (EGFRWT).

Inwardly rectifying potassium (Kir) channels are expressed in many cell types and contribute to a wide range of physiological processes. Particularly, Kir4.1 channels are involved in the astroglial spatial potassium buffering. In this work, we examined the effects of the cationic amphiphilic drug quinacrine on Kir4.1 channels heterologously expressed in HEK293 cells, employing the patch clamp technique. Quinacrine inhibited the currents of Kir4.1 channels in a concentration and voltage dependent manner. In inside-out patches, quinacrine inhibited Kir4.1 channels with an IC50 value of 1.8±0.3μM and with extremely slow blocking and unblocking kinetics. Molecular modeling combined with mutagenesis studies suggested that quinacrine blocks Kir4.1 by plugging the central cavity of the channels, stabilized by the residues E158 and T128. Overall, this study shows that quinacrine blocks Kir4.1 channels, which would be expected to impact the potassium transport in several tissues.

Adaptive optics can correct for optical aberrations. We developed multi-pupil adaptive optics (MPAO), which enables simultaneous wavefront correction over a field of view of 450 × 450 μm2 and expands the correction area to nine times that of conventional methods. MPAO's ability to perform spatially independent wavefront control further enables 3D nonplanar imaging. We applied MPAO to in vivo structural and functional imaging in the mouse brain.

Kir4.1 channels have been implicated in various physiological processes, mainly in the K+ homeostasis of the central nervous system and in the control of glial function and neuronal excitability. Even though, pharmacological research of these channels is very limited. Chloroquine (CQ) is an amino quinolone derivative known to inhibit Kir2.1 and Kir6.2 channels with different action mechanism and binding site. Here, we employed patch-clamp methods, mutagenesis analysis, and molecular modeling to characterize the molecular pharmacology of Kir4.1 inhibition by CQ. We found that this drug inhibits Kir4.1 channels heterologously expressed in HEK-293 cells. CQ produced a fast-onset voltage-dependent pore-blocking effect on these channels. In inside-out patches, CQ showed notable higher potency (IC50 ≈0.5μM at +50mV) and faster onset of block when compared to whole-cell configuration (IC50 ≈7μM at +60mV). Also, CQ showed a voltage-dependent unblock with repolarization. These results suggest that the drug directly blocks Kir4.1 channels by a pore-plugging mechanism. Moreover, we found that two residues (Thr128 and Glu158), facing the central cavity and located within the transmembrane pore, are particularly important structural determinants of CQ block. This evidence was similar to what was previously reported with Kir6.2, but distinct from the interaction site (cytoplasmic pore) CQ-Kir2.1. Thus, our findings highlight the diversity of interaction sites and mechanisms that underlie amino quinolone inhibition of Kir channels.

Potential new EGFRT790M inhibitors comprised of structurally modified diphenylpyrimidine derivatives bearing a morpholine functionality (Mor-DPPYs) were used to improve the activity and selectivity of gefitinib-resistant non-small cell lung cancer (NSCLC) treatment. This led to the identification of inhibitor 10c, which displayed high activity against EGFRT790M/L858R kinase (IC50 = 0.71 nM) and repressed H1975 cell replication harboring EGFRT790M mutations at a concentration of 0.037 μM. Inhibitor 10c demonstrated high selectivity (SI = 631.9) for T790M-containing EGFR mutants over wild type EGFR, suggesting that it will cause less side effects. Moreover, this compound also shows promising antitumor efficacy in a murine EGFRT790M/L858R-driven H1975 xenograft model without affecting body weight. This study provides new potential lead compounds for further development of anti-NSCLC drugs.

This study aimed to clarify that organic anion transporters (OATs) mediate the drug-drug interaction (DDI) between imipenem and cilastatin. After co-administration with imipenem, the plasma concentrations and the plasma concentration-time curve (AUC) of cilastatin were significantly increased, while renal clearance and cumulative urinary excretion of cilastatin were decreased. At the same time, imipenem significantly inhibited the uptake of cilastatin in rat kidney slices and in human OAT1 (hOAT1)-HEK293 and human OAT3 (hOAT3)-HEK293 cells. Probenecid, p-aminohippurate, and benzylpenicillin inhibited the uptake of imipenem and cilastatin in rat kidney slices and in hOAT1- and hOAT3-HEK 293 cells, respectively. The uptakes of imipenem and cilastatin in hOAT1- and hOAT3-HEK 293 cells were significantly higher than that in mock-HEK-293 cells. Moreover, the Km values of cilastatin were increased in the presence of imipenem with unchanged Vmax , indicating that imipenem inhibited the uptake of cilastatin in a competitive manner. When imipenem and cilastatin were co-administered, the level of imipenem was higher compared with imipenem alone both in vivo and in vitro. But, cilastatin significantly inhibited the uptake of imipenem when dehydropeptidase-1 (DPEP1) was silenced by RNAi technology in hOAT1- and hOAT3-HEK 293 cells. In conclusion, imipenem and cilastatin are the substrates of OAT1 and OAT3. OAT1 and OAT3 mediate the DDI between imipenem and cilastatin. Meanwhile, cilastatin also reduces the hydrolysis of imipenem by inhibiting the uptake of imipenem mediated by OAT1 and OAT3 in the kidney as a complement.

Pulmonary fibrosis has been characterized by abnormal proliferation of fibroblasts and massive deposition of the extracellular matrix, which results from a complex interplay of chronic injury and inflammatory responses. MicroRNA-301a (miR-301a) is activated by multiple inflammatory stimulators, contributing to multiple tumorigenesis and autoimmune diseases. This study showed that miR-301a was overexpressed in a bleomycin-induced murine model of pulmonary fibrosis and patients with idiopathic pulmonary fibrosis (IPF). In addition, miR-301a was activated by transforming growth factor β (TGF-β) and interleukin 6 (IL-6) in normal and IPF fibroblasts, which was markedly reversed by the signal transducer and activator of transcription 3 (STAT3) inhibitor. The genetic ablation of miR-301a in mice reduced bleomycin-induced lung fibrosis, and the downregulation of miR-301a restrained proliferation and activation of fibroblasts. Furthermore, this study demonstrated that TSC1 was a functional target of miR-301a in fibroblasts, and the negative regulation of TSC1 by miR-301a promoted the severity of pulmonary fibrosis through the mammalian target of rapamycin (mTOR) signaling pathway. The blocking of miR-301a by the intravenous injection of antagomiR-301a inhibited the proliferation of fibroblasts and the structural destruction of lung tissues in the bleomycin-induced lung fibrosis mouse model. The findings revealed the crucial role of the miR-301a/TSC1/mTOR axis in the pathogenesis of pulmonary fibrosis, suggesting that miR-301a might serve as a potential therapeutic target.

The virtual high-throughput screening (vHTS) approach has been widely used for large database screening to identify potential lead compounds for drug discovery. Due to its high computational demands, docking that allows receptor flexibility has been a challenging problem for virtual screening. Therefore, the selection of protein target conformations is crucial to produce useful vHTS results. Since only a single protein structure is used to screen large databases in most vHTS studies, the main challenge is to reduce false negative rates in selecting compounds for in vitro tests. False negatives are most likely to occur when using apo structures or homology models of protein targets due to the small volume of the binding pocket formed by incorrect side-chain conformations. Even holo protein structures can exhibit high false negative rates due to ligand-induced fit effects, since the shape of the binding pocket highly depends on its bound ligand. To reduce false negative rates and improve success rates for vHTS in drug discovery, we have developed a new Monte Carlo-based approach that optimizes the binding pocket of protein targets. This newly developed Monte Carlo pocket optimization (MCPO) approach was assessed on several datasets showing promising results. The binding pocket optimization approach could be a useful tool for vHTS-based drug discovery, especially in cases when only apo structures or homology models are available.

A novel structural series of quinoline derivatives were designed, synthesized and biologically evaluated as PI3K/mTOR dual inhibitors upon incorporation of C-4 acrylamide fragment. Consequently, all of them exerted remarkable inhibition against PI3Kα with IC50 values ranging from 0.50 to 2.03 nM. Besides, they exhibited sub-micromolar to low micromolar anti-proliferative activity against both prostate cancer PC3 and colorectal cancer HCT116 cell lines. In subsequent profiling, 8i, a representative compound throughout this series, also significantly inhibited other class I PI3Ks and mTOR. In PC3 cells, it remarkably down-regulated the crucial biomarkers of PI3K/Akt/mTOR signaling, including phos-Akt (Ser473), phos-Akt (Thr308), phos-S6 ribosomal protein (Ser235/236), and phos-4E-BP1 (Thr37/46), at a concentration as low as 5 nM. Moreover, 8i displayed favorable metabolic stability with long elimination half-life in both human liver and rat liver microsomes. A further in vivo pharmacokinetic (PK) study demonstrated 8i possessed acceptable oral exposure, peak plasma concentration, and elimination half-life. Taken together, 8i, as a potent PI3K/mTOR dual inhibitor, merited further investigation and structural optimization.

Our goal was to clarify the comparison between elective neck dissection (END) and the wait-and-see policy in neck management for cT1N0 buccal squamous cell carcinoma (SCC).