Elimination of TFA-Mediated Cleavage in Distributed Drug Discovery

Distributed Drug Discovery (D3) is a multi-disciplinary approach to the discovery of new drugs, which target neglected diseases or conditions common to developing-world countries. As part of a continuing effort to improve D3 methodology, two approaches for eliminating the final step TFA-mediated resin cleavage are proposed for investigation. Cleavage under basic conditions (saponification) and mild acid conditions (dilute HCl/hexafluoroisopropanol or dilute HCl/trifluoroethanol) represent improvements in safety and convenience to the undergraduate student researcher. Previous studies have shown that saponification provides yields comparable to the traditional TFA cleavage but recovery is not as convenient. Further improvements in the saponification workup will be evaluated by analyzing the effectiveness of simple trituration with acetone compared to use of a strong anion-exchange resin or drying reagents to isolate the free acid from the salt. Different trituration procedural modifications have been made and are being tested. Results have shown that in the presence of methanol, esterification will occur when the acid is liberated from the salt using HCl. To counter this problem, the samples are first evaporated to remove methanol and then the pH is adjusted with HCl. It was shown that using acetic acid did not result in pH levels low enough to guarantee complete protonation of the carboxylate. Through the use of a Bill-Board, an apparatus that holds six reaction vessels, several procedural modifications can be carried out simultaneously. Analysis is conducted by liquid chromatography coupled with a mass spectrometer and with nuclear magnetic resonance spectroscopy. Further studies will be carried out to assess the efficiency and practicality of using mild acidic conditions for cleavage using HCl/hexafluoroisopropanol or dilute HCl/trifluoroethanol. Both saponification and mild acid cleavage would represent improvements in safety and convenience to the undergraduate student researcher

Saponification of N-Acylated L-Phenylalanine Wang and Merrifield Resins. Assessment of Cleavage Efficiency and Epimerization

poster abstractAs part of a continuing effort to modify Distributed Drug Discovery (D3) synthetic procedures to enhance safety and accommodate the limited resources available to students in developing-world countries, we have recently begun to examine alternatives to trifluoroacetic acid (TFA)-cleavage of amino acid derivatives from polystyrene-based resins. Cleavage of a representative example, N-(4-chlorobenzoyl)-L-phenylalanine, from both Wang and Merrifield resins was accomplished in thirty minutes at room temperature using 0.5M sodium hydroxide in methanol/tetrahydrofuran. In a side-by-side comparison with cleavage using TFA, results indicated that saponification from Wang resin was incomplete after thirty minutes. Experiments designed to examine separately the effect of reaction time, temperature, and concentration were performed and results will be presented. Additionally, investigations were performed to assess the degree of epimerization which had occurred during cleavage of Merrifield-bound L-phenylalanine acylated with both (R)- and (S)-mandelic acid. Results revealed a small but significant amount of epimerization (15:1 to 31:1 diastereomeric ratios) after a thirty-minute cleavage time at room temperature

The MyD88+ phenotype is an adverse prognostic factor in epithelial ovarian cancer

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer

Micrometeoroid Events in LISA Pathfinder

The zodiacal dust complex, a population of dust and small particles that pervades the Solar System, provides important insight into the formation and dynamics of planets, comets, asteroids, and other bodies. Here we present a new set of data obtained using a novel technique: direct measurements of momentum transfer to a spacecraft from individual particle impacts. This technique is made possible by the extreme precision of the instruments flown on the LISA Pathfinder spacecraft, a technology demonstrator for a future space-based gravitational wave observatory that operated near the first Sun-Earth Lagrange point from early 2016 through Summer of 2017. Using a simple model of the impacts and knowledge of the control system, we show that it is possible to detect impacts and measure properties such as the transferred momentum (related to the particle's mass and velocity), direction of travel, and location of impact on the spacecraft. In this paper, we present the results of a systematic search for impacts during 4348 hours of Pathfinder data. We report a total of 54 candidates with momenta ranging from 0.2$\,\mu\textrm{Ns}$ to 230$\,\mu\textrm{Ns}$. We furthermore make a comparison of these candidates with models of micrometeoroid populations in the inner solar system including those resulting from Jupiter-family comets, Oort-cloud comets, Hailey-type comets, and Asteroids. We find that our measured population is consistent with a population dominated by Jupiter-family comets with some evidence for a smaller contribution from Hailey-type comets. This is in agreement with consensus models of the zodiacal dust complex in the momentum range sampled by LISA Pathfinder.Comment: 22 pages, 14 figures, accepted in Ap

Successful Integration of Distributed Drug Discovery (D3) Components: Computational, Synthetic, and Biological Evaluation of Phenylalanine Derivatives as Potential Biofilm Inhibitors

poster abstractDistributed Drug Discovery (D3) is a multidisciplinary approach to identifying molecules that exhibit activity in the treatment of neglected diseases such as malaria, leishmaniasis, and tuberculosis as well as recalcitrant cystic fibrosis (CF) airway infections. D3 seeks to accomplish this task by combining computational chemistry, synthetic chemistry, and biological screening all within an educational framework. Recent reports suggest that D-amino acids are effective in the disassembly and inhibition of bacterial biofilms, which are important for a number of bacterial infections, including those in the CF lung. Utilizing chemical drawing software, we constructed (enumerated) target phenylalanine derivatives from commercially available benzyl halides by substitution at the α position of an amino acid scaffold. A subset of these enumerated molecules was computationally selected for synthesis based on chemical properties. These compounds were synthesized using simple, solid-phase techniques in an undergraduate organic chemistry laboratory class. The resulting racemic unnatural amino acid derivatives were then screened for activity in a biofilm assay. The results show biofilm inhibition with synthesized phenylalanine derivatives. Analysis of the results reveals a trend between lipophilicity and the degree of biofilm inhibition. These new molecules may lead to an avenue for therapy for those CF individuals suffering with bacterial lung infection. As a part of the undergraduate curriculum, this work provides the first example of D3-linked undergraduate student computational analysis, synthesis, and biological evaluation

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death’ (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death’ (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death

Identification of a Novel Risk Locus for Multiple Sclerosis at 13q31.3 by a Pooled Genome-Wide Scan of 500,000 Single Nucleotide Polymorphisms

Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system with an important genetic component and strongest association driven by the HLA genes. We performed a pooling-based genome-wide association study of 500,000 SNPs in order to find new loci associated with the disease. After applying several criteria, 320 SNPs were selected from the microarrays and individually genotyped in a first and independent Spanish Caucasian replication cohort. The 8 most significant SNPs validated in this cohort were also genotyped in a second US Caucasian replication cohort for confirmation. The most significant association was obtained for SNP rs3129934, which neighbors the HLA-DRB/DQA loci and validates our pooling-based strategy. The second strongest association signal was found for SNP rs1327328, which resides in an unannotated region of chromosome 13 but is in linkage disequilibrium with nearby functional elements that may play important roles in disease susceptibility. This region of chromosome 13 has not been previously identified in MS linkage genome screens and represents a novel risk locus for the disease

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

Overexpression of MicroRNAs from the miR-17-92 Paralog Clusters in AIDS-Related Non-Hodgkin's Lymphomas

Individuals infected by HIV are at an increased risk for developing non-Hodgkin's lymphomas (AIDS-NHL). In the highly active antiretroviral therapy (HAART) era, there has been a significant decline in the incidence of AIDS-associated primary central nervous system lymphoma (PCNSL). However, only a modest decrease in incidence has been reported for other AIDS-NHL subtypes. Thus, AIDS-NHLs remain a significant cause of morbidity and mortality in HIV infected individuals. Recently, much attention has been directed toward the role of miRNAs in cancer, including NHL. Several miRNAs, including those encoded by the miR-17-92 polycistron, have been shown to play significant roles in B cell tumorigenesis. However, the role of miRNAs in NHL in the setting of HIV infection has not been defined.We used quantitative realtime PCR to assess the expression of miRNAs from three different paralog clusters, miR-17-92, miR-106a-363, and miR-106b-25 in 24 cases of AIDS-NHLs representing four tumor types, Burkitt's lymphoma (BL, n = 6), diffuse large B-cell lymphoma (DLBCL, n = 8), primary central nervous system lymphoma (PCNSL, n = 5), and primary effusion lymphoma (PEL, n = 5). We also used microarray analysis to identify a differentiation specific miRNA signature of naïve, germinal center, and memory B cell subsets from tonsils (n = 4). miRNAs from the miR-17-92 paralog clusters were upregulated by B cells, specifically during the GC differentiation stage. We also found overexpression of these miRNA clusters in all four AIDS-NHL subtypes. Finally, we also show that select miRNAs from these clusters (miR-17, miR-106a, and miR-106b) inhibited p21 in AIDS-BL and DLBCL cases, thus providing a mechanistic role for these miRNAs in AIDS-NHL pathogenesis.Dysregulation of miR-17-92 paralog clusters is a common feature of AIDS-associated NHLs