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DNA polymerase theta (Polθ) is an attractive synthetic lethal target for drug discovery, predicted to be efficacious against breast and ovarian cancers harboring BRCA-mutant alleles. Here, we describe our hit-to-lead efforts in search of a selective inhibitor of human Polθ (encoded by POLQ). A high-throughput screening campaign of 350,000 compounds identified an 11 micromolar hit, giving rise to the N2-substituted fused pyrazolo series, which was validated by biophysical methods. Structure-based drug design efforts along with optimization of cellular potency and ADME ultimately led to the identification of

In this paper, we report the surface modification of the Li4Ti5O12 (LTO) anode material with a freshly prepared Li-rich PTCLi4 organic molecule using a spray-dryer technique. In addition, burning the resulting powder yielded an electrode material with a few-nanometer-thick carbon coating. For comparison, carbon-coated LTO powder was prepared with graphene oxide (GO) using the same protocol. Organic molecules were first characterized using FTIR, XPS, TGA, XRD, and SEM methods. PTCLi4-coated LTO powders were observed via SEM and the corresponding EDX mapping as well as micro-Raman and XPS spectroscopic analyses confirmed the efficient surface coverage of the anode material. After the burning, a graphitic-like carbon coating with an ID/IG of approximately 0.76 and a thickness of a few nanometers was confirmed by TEM observations. Thermogravimetric analyses revealed that the content of carbon varied from 0.3 to 1.5 wt%, depending on the reaction conditions and material used (i.e., PTCLi4 or GO). Interestingly, electrochemical cycling at 25 °C of PTCLi4-coated LTO electrodes gave rise to superior performance compared to that of the pristine electrode, especially at high C-rates, and carbon-coated electrodes showed intermediate performance. Most importantly, the good cyclability of PTCLi4-coated LTO electrodes was observed with a specific capacity of 145 mA h g−1 after 100 cycles at a C/2 rate with an average coulombic efficiency of 100%. XPS analyses performed on aged electrodes revealed a low degradation of the electrolyte with a lower concentration of LiF on the surface of the PTCLi4-coated LTO electrodes. Finally, the cycling of LTO electrodes demonstrated the potential of using the PTCLi4 coating to increase the Li-ion transfer at the electrode–electrolyte interface at subzero temperatures. In fact, the PTCLi4-coated LTO electrode delivered almost the same specific capacity at a C/2 rate when cycled at –20 °C as the pristine electrode cycled at 25 °C.

The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, since the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here, we present the genetic interaction profiles of APE2-, APE1- and TDP1-deficiency coupled to a biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3′ DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3′-blocking lesions relevant to the APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3′ blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.

We report the structure and morphology of the solid-solution series LiMnyFe1EyPO4 isomorphous with olivine synthesized by the hydrothermal route. Experimental studies include combined characterizations such as XRD, FTIR, SEM and HRTEM. Carbon-coated nanoparticles were prepared by the lactose route, which quality was investigated by Raman spectroscopy.

We report the electrochemical properties of LiMnyFe1-yPO4 (0.5≤y≤0.8) cathode materials synthesized by hydrothermal route. Special attention was taken to optimize the carbon deposit on the surface of particles in order to make easy comparison of the electrochemical performance as a function composition with the same particle size. The substitution led to the modification of the electrochemical performance, such as initial capacity, capacity fading and polarization. We present the modified Peukert diagrams for C-rate in the range from C/12 to 40C.

A novel series of EP(4) ligands, based on a benzyl indoline scaffold, has been discovered. It was found that agonism and antagonism in this series can be easily modulated by minor modifications on the benzyl group. The pharmacokinetic, metabolic and pharmacological profiles of these compounds was explored. It was found that these compounds show good pharmacokinetics in rat and are efficacious in pre-clinical models of pain and inflammation.

Neuropeptide S (NPS) and its receptor (NPSR) are thought to have a role in asthma pathogenesis; a number of single nucleotide polymorphisms within NPSR have been shown to be associated with an increased prevalance of asthma. One such single nucleotide polymorphism leads to the missense mutation N107I, which results in an increase in the potency of NPS for NPSR. To gain insight into structure-function relationships within NPS and NPSR, we first carried out a limited structural characterization of NPS and subjected the peptide to extensive mutagenesis studies. Our results show that the NH2-terminal third of NPS, in particular residues Phe-2, Arg-3, Asn-4, and Val-6, are necessary and sufficient for activation of NPSR. Furthermore, part of a nascent helix within the peptide, spanning residues 5 through 13, acts as a regulatory region that inhibits receptor activation. Notably, this inhibition is absent in the asthma-linked N107I variant of NPSR, suggesting that residue 107 interacts with the aforementioned regulatory region of NPS. Whereas this interaction may be at the root of the increase in potency associated with the N107I variant, we show here that the mutation also causes an increase in cell-surface expression of the mutant receptor, leading to a concomitant increase in the maximal efficacy (Emax) of NPS. Our results identify the key residues of NPS involved in NPSR activation and suggest a molecular basis for the functional effects of the N107I mutation and for its putative pathophysiological link with asthma.

A novel indole series of PGD2 receptor (DP receptor) antagonists is presented. Optimization of this series led to the identification of potent and selective DP receptor antagonists. In particular, antagonists 35 and 36 were identified with Ki values of 2.6 and 1.8 nM, respectively. These two antagonists are also potent in a DP functional assay where they inhibit the PGD2 induced cAMP production in platelet rich plasma with IC50 values of 7.9 and 8.6 nM, respectively. The structure–activity relationships of this indole series of DP receptor antagonists will also be discussed.

Background Oxidative Stress contributes to the pathogenesis of many diseases. The NRF2/KEAP1 axis is a key transcriptional regulator of the anti-oxidant response in cells. Nrf2 knockout mice have implicated this pathway in regulating inflammatory airway diseases such as asthma and COPD. To better understand the role the NRF2 pathway has on respiratory disease we have taken a novel approach to define NRF2 dependent gene expression in a relevant lung system. Methods Normal human lung fibroblasts were transfected with siRNA specific for NRF2 or KEAP1. Gene expression changes were measured at 30 and 48 hours using a custom Affymetrix Gene array. Changes in Eotaxin-1 gene expression and protein secretion were further measured under various inflammatory conditions with siRNAs and pharmacological tools. Results An anti-correlated gene set (inversely regulated by NRF2 and KEAP1 RNAi) that reflects specific NRF2 regulated genes was identified. Gene annotations show that NRF2-mediated oxidative stress response is the most significantly regulated pathway, followed by heme metabolism, metabolism of xenobiotics by Cytochrome P450 and O-glycan biosynthesis. Unexpectedly the key eosinophil chemokine Eotaxin-1/CCL11 was found to be up-regulated when NRF2 was inhibited and down-regulated when KEAP1 was inhibited. This transcriptional regulation leads to modulation of Eotaxin-1 secretion from human lung fibroblasts under basal and inflammatory conditions, and is specific to Eotaxin-1 as NRF2 or KEAP1 knockdown had no effect on the secretion of a set of other chemokines and cytokines. Furthermore, the known NRF2 small molecule activators CDDO and Sulphoraphane can also dose dependently inhibit Eotaxin-1 release from human lung fibroblasts. Conclusions These data uncover a previously unknown role for NRF2 in regulating Eotaxin-1 expression and further the mechanistic understanding of this pathway in modulating inflammatory lung disease.

Two new series of EP 4 antagonists based on naphthalene/quinoline scaffolds have been identified as part of our on-going efforts to develop treatments for inflammatory pain. One series contains an acidic sulfonylurea pharmacophore, whereas the other is a neutral amide. Both series show subnanomolar intrinsic binding potency towards the EP 4 receptor, and excellent selectivity towards other prostanoid receptors. While the amide series generally displays poor pharmacokinetic parameters, the sulfonylureas exhibit greatly improved profile. MF - 592 , the optimal compound from the sulfonylurea series, has a desirable overall preclinical profile that suggests it is suitable for further development.

The discovery of highly potent and selective second generation EP(4) antagonist MK-2894 (34d) is discussed. This compound exhibits favorable pharmacokinetic profile in a number of preclinical species and potent anti-inflammatory activity in several animal models of pain/inflammation. It also shows favorable GI tolerability profile in rats when compared to traditional NSAID indomethacin.

Azaindole based structures were evaluated as DP1 receptor antagonists. This work has lead to the discovery of potent, selective and distinct DP1 receptor antagonists.

Interleukin 13 (IL-13) is considered to be a key driver of the development of airway allergic inflammation and remodeling leading to airway hyperresponsiveness (AHR). How precisely IL-13 leads to the development of airway inflammation, AHR, and mucus production is not fully understood. In order to identify key mediators downstream of IL-13, we administered adenovirus IL-13 to specifically induce IL-13-dependent inflammation in the lungs of mice. This approach was shown to induce cardinal features of lung disease, specifically airway inflammation, elevated cytokines, AHR, and mucus secretion. Notably, the model is resistant to corticosteroid treatment and is characterized by marked neutrophilia, two hallmarks of more severe forms of asthma. To identify IL-13-dependent mediators, we performed a limited-scale two-dimensional SDS-PAGE proteomic analysis and identified proteins significantly modulated in this model. Intriguingly, several identified proteins were unique to this model, whereas others correlated with those modulated in a mouse ovalbumin-induced pulmonary inflammation model. We corroborated this approach by illustrating that proteomic analysis can identify known pathways/mediators downstream of IL-13. Thus, we have characterized a murine adenovirus IL-13 lung model that recapitulates specific disease traits observed in human asthma, and have exploited this model to identify effectors downstream of IL-13. Collectively, these findings will enable a broader appreciation of IL-13 and its impact on disease pathways in the lung.

A new series of indole-based antagonists of the PGD(2) receptor subtype 1 (DP1 receptor) was identified and the progress of the structure-activity relationship study to the identification of potent and selective antagonists is presented. Selective DP1 antagonists with high potency and selectivity were prepared. Of particular interest is the DP1 antagonist 26 with a K(i) value of 1 nM for the DP1 receptor and an IC(50) value of 4.6 nM in a DP1 functional assay for the inhibition of the PGD(2) induced cAMP production in platelet rich plasma (PRP).

A new series of EP4 antagonists based on a quinoline acylsulfonamide scaffold have been identified as part of our on-going efforts to develop treatments for chronic inflammation. These compounds show subnanomolar intrinsic binding potency towards the EP4 receptor, and excellent selectivity towards other prostanoid receptors. Acceptable pharmacokinetic profiles have also been demonstrated across a series of preclinical species.

The discovery of the potent and selective prostaglandin D2 (PGD2) receptor (DP) antagonist [(3R)-4-(4-chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]-acetic acid (13) is presented. Initial lead antagonists 6 and 7 were found to be potent and selective DP antagonists (DP Ki = 2.0 nM for each); however, they both suffered from poor pharmacokinetic profiles, short half-lives and high clearance rates in rats. Rat bile duct cannulation studies revealed that high concentrations of parent drug were present in the biliary fluid (Cmax = 1100 microM for 6 and 3900 microM for 7). This pharmacokinetic liability was circumvented by replacing the 7-methylsulfone substituent present in 6 and 7 with a fluorine atom resulting in antagonists with diminished propensity for biliary excretion and with superior pharmacokinetic profiles. Further optimization led to the discovery of the potent and selective DP antagonist 13.

Neuropeptide S (NPS) and its receptor (NPSR) are thought to have a role in asthma pathogenesis; a number of single nucleotide polymorphisms within NPSR have been shown to be associated with an increased prevalance of asthma. One such single nucleotide polymorphism leads to the missense mutation N107I, which results in an increase in the potency of NPS for NPSR. To gain insight into structure-function relationships within NPS and NPSR, we first carried out a limited structural characterization of NPS and subjected the peptide to extensive mutagenesis studies. Our results show that the NH(2)-terminal third of NPS, in particular residues Phe-2, Arg-3, Asn-4, and Val-6, are necessary and sufficient for activation of NPSR. Furthermore, part of a nascent helix within the peptide, spanning residues 5 through 13, acts as a regulatory region that inhibits receptor activation. Notably, this inhibition is absent in the asthma-linked N107I variant of NPSR, suggesting that residue 107 interacts with the aforementioned regulatory region of NPS. Whereas this interaction may be at the root of the increase in potency associated with the N107I variant, we show here that the mutation also causes an increase in cell-surface expression of the mutant receptor, leading to a concomitant increase in the maximal efficacy (E(max)) of NPS. Our results identify the key residues of NPS involved in NPSR activation and suggest a molecular basis for the functional effects of the N107I mutation and for its putative pathophysiological link with asthma.

The anaphylatoxin C3a is an important immune regulator with a number of distinct functions in both innate and adaptive immunity. Many of these roles have been ascribed to C3a based on studies in mice genetically modified to lack its precursor, C3, or its receptor, C3aR. However, other presumed functions of C3a are based on results obtained with a recently described small molecule ligand of C3aR, SB 290157. Although this compound was originally described as an antagonist and appears to act as such in some systems, it has recently been shown to have effects that cannot be explained by simple antagonism of C3aR. In the current study, SB 290157 is shown to have full agonist activity on C3aR in a variety of cell systems, including a calcium mobilization assay in transfected RBL cells, a β-lactamase assay in CHO-NFAT-bla-Gα16 cells and an enzyme-release assay in differentiated U-937 cells. On the other hand, the compound lacks agonist activity in guinea pig platelets, cells known to express C3aR at very low levels. SB 290157 agonism of C3aR is consistent with recent discrepant data obtained using this molecule. These results caution against attributing novel roles to C3a based on data obtained with SB 290157 and highlight a continuing need for the identification of true small molecule C3aR antagonists.

Analogues of PGE(2) wherein the hydroxycyclopentanone ring has been replaced by a lactam have been prepared and evaluated as ligands for the EP(4) receptor. An optimized compound (19a) shows high potency and agonist efficacy at the EP(4) receptor and is highly selective over the other seven known prostaglandin receptors.

The mouse multidrug resistance gene family consists of three genes (mdr1, mdr2, and mdr3) encoding P-glycoprotein. We show that the expression of mdr1 is increased at the transcriptional level upon treatment of the hepatoma cell line Hepa-1c1c7 with the polycyclic aromatic hydrocarbon 3-methylcholanthrene (3-MC). This increase is not observed in the aromatic hydrocarbon receptor (AhR)-defective TAOc1BP(r)c1 and the AhR nuclear translocator (Arnt)-defective BP(r)c1 variants, demonstrating that the induction of mdr1 by 3-MC requires AhR.Arnt. We show that the mdr1 promoter (-1165 to +84) is able to activate the expression of a reporter gene in response to 3-MC in Hepa-1c1c7 but not in BP(r)c1 cells. Deletion analysis indicated that the region from -245 to -141 contains cis-acting sequences mediating the induction, including a potential p53 binding sequence. 3-MC treatment of the cells increased the levels of p53 and induced p53 binding to the mdr1 promoter in an AhR.Arnt-dependent manner. Mutations in the p53 binding site abrogated induction of mdr1 by 3-MC, indicating that p53 binding to the mdr1 promoter is essential for the induction. Benzo(a)pyrene, a polycyclic aromatic hydrocarbon and AhR ligand, which, like 3-MC, is oxidized by metabolizing enzymes regulated by AhR.Arnt, also activated p53 and induced mdr1 transcription. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, an AhR ligand resistant to metabolic breakdown, had no effect. These results indicate that the transcriptional induction of mdr1 by 3-MC and benzo(a)pyrene is directly mediated by p53 but that the metabolic activation of these compounds into reactive species is necessary to trigger p53 activation. The ability of the anticancer drug and potent genotoxic agent daunorubicin to induce mdr1 independently of AhR.Arnt further supports the proposition that mdr1 is transcriptionally up-regulated by p53 in response to DNA damage.

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An optimized analog of prostaglandin E 2 which incorporates a γ-lactam in place of the cyclopentanone ring and which incorporates metabolically stabilized side chains, has been identified and shown to exhibit potent and selective EP4 receptor agonism. The compound (2) (L-000902688) is also well absorbed on oral dosing and exhibits a long half-life making it an excellent tool for the study of the role of EP4 receptor in physiology and disease. An efficient synthesis of 2 from a chiral synthon is described.