A New Drug Discovery Platform: Application to DNA Polymerase Eta and Apurinic/Apyrimidinic Endonuclease 1.

The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM ("SAR by X-ray Poses Quickly") platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or "undruggable" targets, allows for (i) hit generation; (ii) the mapping of protein-ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.

Be Responsible?

The bystander effect reveals that people are less likely to help a person in need when others are present. We examined the impact of priming the concept of responsibility on the bystander effect in a field study. Lone pedestrians (N = 259) were randomly assigned to a two (Bystanders: none and three nonresponsive bystanders) by two (Shirt: blank shirt and shirt with "Be Responsible" written on the front) design. A researcher dropped eight pens approximately 15 ft from a lone pedestrian, while wearing one of the two shirts in the presence/absence of bystanders (confederates). The bystander effect was found: Pedestrians helped pick up pens more frequently in the no bystanders condition (59.05% helped) compared to the nonresponsive bystanders condition (41.67% helped). The responsibility prime tended to boost helping rates, but it did not significantly increase helping rates either as a main effect or as part of an interaction term. The bystander effect was replicated in a field setting, but priming the concept of responsibility did not appear to reduce it.

Immature teratoma in an adolescent with Proteus syndrome: A novel association.

Proteus syndrome (PS) is a complex disorder characterized by variable clinical findings of overgrowth and tumor susceptibility. This report presents the first known association between PS and an ovarian germ cell tumor in an adolescent with immature teratoma. A review of the diagnosis of PS and associated tumors is included.

How old is too old? In vivo engraftment of human peripheral blood stem cells cryopreserved for up to 18 years - implications for clinical transplantation and stability programs.

BACKGROUND: Peripheral blood stem cells (PBSC) are commonly cryopreserved awaiting clinical use for hematopoietic stem cell transplant. Long term cryopreservation is commonly defined as five years or longer, and limited data exists regarding how long PBSC can be cryopreserved and retain the ability to successfully engraft. Clinical programs, stem cell banks, and regulatory and accrediting agencies interested in product stability would benefit from such data. Thus, we assessed recovery and colony forming ability of PBSC following long-term cryopreservation as well as their ability to engraft in NOD/SCID/IL-2Rγnull (NSG) mice. AIM: To investigate the in vivo engraftment potential of long-term cryopreserved PBSC units. METHODS: PBSC units which were collected and frozen using validated clinical protocols were obtained for research use from the Cellular Therapy Laboratory at Indiana University Health. These units were thawed in the Cellular Therapy Laboratory using clinical standards of practice, and the pre-freeze and post-thaw characteristics of the units were compared. Progenitor function was assessed using standard colony-forming assays. CD34-selected cells were transplanted into immunodeficient mice to assess stem cell function. RESULTS: Ten PBSC units with mean of 17 years in cryopreservation (range 13.6-18.3 years) demonstrated a mean total cell recovery of 88% ± 12% (range 68%-110%) and post-thaw viability of 69% ± 17% (range 34%-86%). BFU-E growth was shown in 9 of 10 units and CFU-GM growth in 7 of 10 units post-thaw. Immunodeficient mice were transplanted with CD34-selected cells from four randomly chosen PBSC units. All mice demonstrated long-term engraftment at 12 wk with mean 34% ± 24% human CD45+ cells, and differentiation with presence of human CD19+, CD3+ and CD33+ cells. Harvested bone marrow from all mice demonstrated growth of erythroid and myeloid colonies. CONCLUSION: We demonstrated engraftment of clinically-collected and thawed PBSC following cryopreservation up to 18 years in NSG mice, signifying likely successful clinical transplantation of PBSC following long-term cryopreservation.

Laser Patterned Polymer/Carbon Nanotubes Composite Electrodes for Flexible Silicon Nanowire Transistors.

Fabrication techniques such as laser patterning offer excellent potential for low cost and large area device fabrication. Conductive polymers can be used to replace expensive metallic inks such as silver and gold nanoparticles for printing technology. Electrical conductivity of the polymers can be improved by blending with carbon nanotubes. In this work, formulations of acid functionalized multiwalled carbon nanotubes (f-MWCNTs) and poly(ethylenedioxythiophene) [PEDOT]:polystyrene sulphonate [PSS] were processed, and thin films were prepared on plastic substrates. Conductivity of PEDOT:PSS increased almost four orders of magnitude after adding f-MWCNTs. Work function of PEDOT:PSS/f-MWCNTs films was ∼0.5 eV higher as compared to the work function of pure PEDOT:PSS films, determined by Kelvin probe method. Field-effect transistors source-drain electrodes were prepared on PET plastic substrates where PEDOT:PSS/f-MWCNTs were patterned using laser ablation at 44 mJ/pulse energy to define 36 µm electrode separation. Silicon nanowires were deposited using dielectrophoresis alignment technique to bridge laser patterned electrodes. Top-gated nanowire field effect transistors were completed by depositing parylene C as polymer gate dielectric and gold as the top-gate electrode. Transistor characteristics showed p-type conduction with excellent gate electrode coupling, with an ON/OFF ratio of ∼200. Thereby, we demonstrate the feasibility of using high workfunction, printable PEDOT:PSS/f-MWCNTs composite inks for laser patterned source/drain electrodes for nanowire transistors on flexible substrates.

Correction: Conformationally restricted benzothienoazepine respiratory syncytial virus inhibitors: their synthesis, structural analysis and biological activities.

[This corrects the article DOI: 10.1039/C8MD00033F.].

Conformationally restricted benzothienoazepine respiratory syncytial virus inhibitors: their synthesis, structural analysis and biological activities.

Atropisomeric drug substances are known to have different biological properties. Compounds containing the N-benzoylbenzazepine motif have been shown to exhibit energetically restricted rotation around the Ar(CO)N axis. Herein we report, for the first time, the synthesis, physical characterisation and anti-viral profiles of a series of C-4 and C-5 methylated thieno-benzazepines. NMR analysis reveals that incorporation of a single additional substituent at either of these loci influences the conformational dynamics of the azepine ring system. In the case of the C-5 alkyl analogues, the influence of the new stereocentre is so pronounced that its absolute configuration determines which unique atropisomer is obtained following the generation of the benzazepine nucleus. Screening of the alkylated derivatives for their anti-respiratory syncytial virus (RSV) activity indicates that the desired viral pathogenicity is strongly associated with the conformation adopted by the modified tricyclic scaffolds. This is particularly evident in the case of the C-5 homologues in which one atropisomer was found to be potently active and the other essentially inert. These results provide compelling evidence that we have determined the bioactive conformation shared by RSV inhibitors that employ the thienobenazapine nucleus as their core molecular architecture. Furthermore, the understanding obtained from these studies may make it possible to design improved agents against RSV infection in the future.

Synthesis and Activity of a Novel Autotaxin Inhibitor-Icodextrin Conjugate.

Autotaxin is an extracellular phospholipase D that catalyzes the hydrolysis of lysophosphatidyl choline (LPC) to generate the bioactive lipid lysophosphatidic acid (LPA). Autotaxin has been implicated in many pathological processes relevant to cancer. Intraperitoneal administration of an autotaxin inhibitor may benefit patients with ovarian cancer; however, low molecular mass compounds are known to be rapidly cleared from the peritoneal cavity. Icodextrin is a polymer that is already in clinical use because it is slowly eliminated from the peritoneal cavity. Herein we report conjugation of the autotaxin inhibitor HA155 to icodextrin. The conjugate inhibits autotaxin activity (IC50 = 0.86 ± 0.13 µg mL-1) and reduces cell migration. Conjugation of the inhibitor increased its solubility, decreased its membrane permeability, and improved its intraperitoneal retention in mice. These observations demonstrate the first application of icodextrin as a covalently-bonded drug delivery platform with potential use in the treatment of ovarian cancer.

In Vitro and In Vivo Efficacy of a Novel and Long-Acting Fungicidal Azole, PC1244, on Aspergillus fumigatus Infection.

The antifungal effects of the novel triazole PC1244, designed for topical or inhaled administration, against Aspergillus fumigatus were tested in a range of in vitro and in vivo studies. PC1244 demonstrated potent antifungal activities against clinical A. fumigatus isolates (n = 96) with a MIC range of 0.016 to 0.25 µg/ml, whereas the MIC range for voriconazole was 0.25 to 0.5 µg/ml. PC1244 was a strong tight-binding inhibitor of recombinant A. fumigatus CYP51A and CYP51B (sterol 14α-demethylase) enzymes and strongly inhibited ergosterol synthesis in A. fumigatus with a 50% inhibitory concentration of 8 nM. PC1244 was effective against a broad spectrum of pathogenic fungi (MIC range, <0.0078 to 2 µg/ml), especially Aspergillus terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, Cryptococcus gattii, Cryptococcus neoformans, and Rhizopus oryzae PC1244 also proved to be quickly absorbed into both A. fumigatus hyphae and bronchial epithelial cells, producing persistent antifungal effects. In addition, PC1244 showed fungicidal activity (minimum fungicidal concentration, 2 µg/ml) which indicated that it was 8-fold more potent than voriconazole. In vivo, once-daily intranasal administration of PC1244 (3.2 to 80 µg/ml) to temporarily neutropenic, immunocompromised mice 24 h after inoculation with itraconazole-susceptible A. fumigatus substantially reduced the fungal load in the lung, the galactomannan concentration in serum, and circulating inflammatory cytokine levels. Furthermore, 7 days of extended prophylaxis with PC1244 showed in vivo effects superior to those of 1 day of prophylactic treatment, suggesting accumulation of the effects of PC1244. Thus, PC1244 has the potential to be a novel therapy for the treatment of A. fumigatus infection in the lungs of humans.

Discovery of novel benzothienoazepine derivatives as potent inhibitors of respiratory syncytial virus.

The development of novel non-nucleoside inhibitors of the RSV polymerase complex is of significant clinical interest. Compounds derived from the benzothienoazepine core, such as AZ-27, are potent inhibitors of RSV viruses of the A-subgroup, but are only moderately active against the B serotype and as yet have not demonstrated activity in vivo. Herein we report the discovery of several novel families of C-2 arylated benzothienoazepine derivatives that are highly potent RSV polymerase inhibitors and reveal an exemplary structure, compound 4a, which shows low nanomolar activity against both RSV A and B viral subtypes. Furthermore, this compound is effective at suppressing viral replication, when administered intranasally, in a rodent model of RSV infection. These results suggest that compounds belonging to this chemotypes have the potential to provide superior anti-RSV agents than those currently available for clinical use.

In Vitro and In Vivo Antifungal Profile of a Novel and Long-Acting Inhaled Azole, PC945, on Aspergillus fumigatus Infection.

The profile of PC945, a novel triazole antifungal designed for administration via inhalation, was assessed in a range of in vitro and in vivo studies. PC945 was characterized as a potent, tightly binding inhibitor of Aspergillus fumigatus sterol 14α-demethylase (CYP51A and CYP51B) activity (50% inhibitory concentrations [IC50s], 0.23 µM and 0.22 µM, respectively) with characteristic type II azole binding spectra. Against 96 clinically isolated A. fumigatus strains, the MIC values of PC945 ranged from 0.032 to >8 µg/ml, while those of voriconazole ranged from 0.064 to 4 µg/ml. Spectrophotometric analysis of the effects of PC945 against itraconazole-susceptible and -resistant A. fumigatus growth yielded IC50 (determined based on optical density [OD]) values of 0.0012 to 0.034 µg/ml, whereas voriconazole (0.019 to >1 µg/ml) was less effective than PC945. PC945 was effective against a broad spectrum of pathogenic fungi (with MICs ranging from 0.0078 to 2 µg/ml), including Aspergillus terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, Cryptococcus gattii, Cryptococcus neoformans, and Rhizopus oryzae (1 or 2 isolates each). In addition, when A. fumigatus hyphae or human bronchial cells were treated with PC945 and then washed, PC945 was found to be absorbed quickly into both target and nontarget cells and to produce persistent antifungal effects. Among temporarily neutropenic immunocompromised mice infected with A. fumigatus intranasally, 50% of the animals survived until day 7 when treated intranasally with PC945 at 0.56 µg/mouse, while posaconazole showed similar effects (44%) at 14 µg/mouse. This profile affirms that topical treatment with PC945 should provide potent antifungal activity in the lung.

Discovery of Narrow Spectrum Kinase Inhibitors: New Therapeutic Agents for the Treatment of COPD and Steroid-Resistant Asthma.

The discovery of a novel series of therapeutic agents that has been designed and optimized for treating chronic obstructive pulmonary disease is reported. The pharmacological strategy was based on the identification of compounds that inhibit a defined subset of kinase enzymes modulating inflammatory processes that would be effective against steroid refractory disease and exhibit a sustained duration of action after inhaled delivery.

Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction.

Cystic fibrosis is mostly caused by the F508del mutation, which impairs CFTR protein from exiting the endoplasmic reticulum due to misfolding. VX-809 is a small molecule that rescues F508del-CFTR localization, which recently went into clinical trial but with unknown mechanism of action (MoA). Herein, we assessed if VX-809 is additive or synergistic with genetic revertants of F508del-CFTR, other correctors, and low temperature to determine its MoA. We explored and integrated those various agents in combined treatments, showing how they add to each other to identify their complementary MoA upon correction of F508del-CFTR. Our experimental and modeling data, while compatible with putative binding of VX-809 to NBD1:ICL4 interface, also indicate scope for further synergistic F508del-CFTR correction by other compounds at distinct conformational sites/cellular checkpoints, thus suggesting requirement of combined therapies to fully rescue F508del-CFTR.

Plant-derived medicines: a novel class of immunological adjuvants.

Plant-derived medicines have a long history of use for the prevention and treatment of human disease. Today, many pharmaceuticals currently approved by the Food and Drug Administration (FDA) have origins to plant sources. A major role for plant-derived compounds based on the reported immunomodulatory effects has emerged in recent times and has led to the rigorous scientific examination to determine efficacy and safety. The discovery of novel plant compounds with immune system modulating activities has become an increasingly important area of research, particularly in the search for new-generation vaccine adjuvants. This review discusses the important role of plant-derived medicines as immunomodulators and provides evidence in support of the continued investigation of this new class of drugs for the maintenance of human health. The identification and characterization of plant compounds that augment new or existing vaccines, and in particular mucosally administered vaccines, will be of significant interest to vaccinologists and immunologists.

A novel foregut mucin characterized by a murine monoclonal autoantibody.

Autoantibodies to gastric cellular antigens and glycoproteins including mucins and Lewis X and Y antigens have been implicated in the induction of autoimmune gastritis. Monoclonal antibody D10 (D10 MAb) recognizes a highly conserved mucin expressed in the foregut of mammals and other vertebrates. The objective of this study was to biochemically characterize the autoantigen identified by D10 MAb and examine its autoimmunogenicity in the mouse. Characterization of the mucin autoantigen was undertaken following purification, by amino acid and carbohydrate analyses, deglycosylation, SDS-PAGE, and immunoblotting using D10 MAb. Autoimmune reactivity and specificity of D10 MAb were validated by immunohistochemistry and ELISA using mouse tissue. Induction of autoimmune gastritis was investigated following immunization of mice with D10 MAb-reactive heterologous mucin. D10 MAb was shown to be a murine anti-mucin autoantibody with a unique pattern of immunohistochemical staining of Brunner's glands of the duodenum and the cardiac glands, mucous neck cells, and pyloric glands of the stomach from inbred Balb/c mice in patterns identical to that previously reported in human tissue. Amino acid and carbohydrate analysis of purified D10 mucin reflected a compositional profile of a typical mucin molecule. Confirmation that D10 MAb recognizes a mucin was also provided by demonstration that the carbohydrate epitope resides on a high molecular weight (>1x10(6)Da), high-density (>1.40 g/mL) molecule comprised of greater than 60% carbohydrate. Mice immunized with D10 MAb-reactive, purified, heterologous mucin produced autoantibodies of identical specificity to the original D10 MAb. These data demonstrate the autoimmunogenic properties of a novel foregut mucin and raise the potential of anti-mucin autoantibodies in the induction of autoimmune gastritis.

Manganese chelation therapy extends survival in a mouse model of M1000 prion disease.

Previous in vitro and in vivo investigations have suggested manganese (Mn(2+)) may play a role in pathogenesis through facilitating refolding of the normal cellular form of the prion protein into protease resistant, pathogenic isoforms (PrP(Sc)), as well as the subsequent promotion of higher order aggregation of these abnormal conformers. To further explore the role of Mn(2+) in pathogenesis, we undertook a number of studies, including an assessment of the disease modifying effects of chelation therapy in a well-characterized mouse model of prion disease. The di-sodium, calcium derivative of the chelator, cyclohexanediaminetetraacetic acid (Na(2)CaCDTA), was administered intraperitoneally to mice inoculated intra-cerebrally with either high or low-dose inocula, with treatment beginning early (shortly after inoculation) or late (at the usual mid-survival point of untreated mice). Analyses by inductively coupled plasma-mass spectrometry demonstrated brain Mn(2+) levels were selectively reduced by up to 50% in treated mice compared with untreated controls, with copper, iron, zinc and cobalt levels unchanged. In mice administered high-dose inocula, none of the treatment groups displayed an increase in survival although western blot analyses of early intensively treated mice showed reduced brain PrP(Sc) levels; mice infected using low-dose inocula however, showed a significant prolongation of survival (p = 0.002). Although our findings support a role for Mn(2+) in prion disease, further studies are required to more precisely delineate the extent of pathogenic involvement.

Identification of a novel vaccine adjuvant that stimulates and maintains diphtheria toxoid immunity.

Immunomodulation by plant-derived medicines is well-documented with effects on both innate and adaptive immunity. This study reports potent and long-lasting diphtheria toxoid-specific immunity by the botanical medicinal, Rehmannia Six Formula, using an in vivo mouse model of vaccine immunity. A significant vaccine adjuvant effect was observed with an increase in serum anti-diphtheria toxoid total and IgG antibodies following oral administration of Rehmannia Six Formula to mice. This response was antigen-specific and was still detectable six months following botanical medicinal treatment, suggesting that Rehmannia Six Formula could help maintain protective antibody levels in populations where vaccine coverage is low. Rehmannia Six Formula was well-tolerated with no adverse effects on mouse weight or survival observed in this study and suggests a potential role as a novel vaccine adjuvant preparation.

Stimulation of tetanus toxoid-specific immune responses by a traditional Chinese herbal medicine.

The immunomodulatory properties of botanical medicinals are well-documented. In this study, the capacity for the traditional Chinese herbal medicine, Rehmannia Six Formula (R6F), to stimulate anti-tetanus toxoid (TT) immunity following oral administration to mice was examined. A significant rise in serum anti-TT antibody levels were observed in R6F-treated mice immunized with a minimum immunogenic dose of 10 microg TT suggesting an oral adjuvant effect. No such enhancement was found for unimmunized mice treated with R6F. This anti-TT response was preferentially IgG and antigen-specific in relation to antibody reactivity to a panel of unrelated antigens. The R6F used was safe with no adverse effects on mouse weight or survival, providing evidence for the use of R6F as an oral adjuvant.