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The role that broadly neutralizing antibodies (bNAbs) play in natural clearance of human hepatitis C virus (HCV) infection and the underlying mechanisms remain unknown. Here, we investigate the mechanism by which bNAbs, isolated from two humans who spontaneously cleared HCV infection, contribute to HCV control. Using viral gene sequences amplified from longitudinal plasma of the two subjects, we found that these bNAbs, which target the front layer of the HCV envelope protein E2, neutralized most autologous HCV strains. Acquisition of resistance to bNAbs by some autologous strains was accompanied by progressive loss of E2 protein function, and temporally associated with HCV clearance. These data demonstrate that bNAbs can mediate clearance of human HCV infection by neutralizing infecting strains and driving escaped viruses to an unfit state. These immunopathologic events distinguish HCV from HIV-1 and suggest that development of an HCV vaccine may be achievable.
Copyright © 2018 Elsevier Inc. All rights reserved.
Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.
Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
This letter describes the chemical optimization of a new series of M positive allosteric modulators (PAMs) based on a novel benzomorpholine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0486846 (7), devoid of adverse effect liability. This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0486846 and details all of the challenges faced in allosteric modulator programs (both steep and flat SAR, as well as subtle structural changes affecting CNS penetration and overall physiochemical and DMPK properties).
Copyright © 2018 Elsevier Ltd. All rights reserved.
During reduced energy intake, skeletal muscle maintains homeostasis by rapidly suppressing insulin-stimulated glucose utilization. Loss of this adaptation is observed with deficiency of the fatty acid transporter CD36. A similar loss is also characteristic of the insulin-resistant state where CD36 is dysfunctional. To elucidate what links CD36 to muscle glucose utilization, we examined whether CD36 signaling might influence insulin action. First, we show that CD36 deletion specific to skeletal muscle reduces expression of insulin signaling and glucose metabolism genes. It decreases muscle ceramides but impairs glucose disposal during a meal. Second, depletion of CD36 suppresses insulin signaling in primary-derived human myotubes, and the mechanism is shown to involve functional CD36 interaction with the insulin receptor (IR). CD36 promotes tyrosine phosphorylation of IR by the Fyn kinase and enhances IR recruitment of P85 and downstream signaling. Third, pretreatment for 15 min with saturated fatty acids suppresses CD36-Fyn enhancement of IR phosphorylation, whereas unsaturated fatty acids are neutral or stimulatory. These findings define mechanisms important for muscle glucose metabolism and optimal insulin responsiveness. Potential human relevance is suggested by genome-wide analysis and RNA sequencing data that associate genetically determined low muscle CD36 expression to incidence of type 2 diabetes.
© 2018 by the American Diabetes Association.
Selective activation of the M subtype of muscarinic acetylcholine receptor, via positive allosteric modulation (PAM), is an exciting strategy to improve cognition in schizophrenia and Alzheimer's disease patients. However, highly potent M ago-PAMs, such as MK-7622, PF-06764427, and PF-06827443, can engender excessive activation of M, leading to agonist actions in the prefrontal cortex (PFC) that impair cognitive function, induce behavioral convulsions, and result in other classic cholinergic adverse events (AEs). Here, we report a fundamentally new and highly selective M PAM, VU0486846. VU0486846 possesses only weak agonist activity in M-expressing cell lines with high receptor reserve and is devoid of agonist actions in the PFC, unlike previously reported ago-PAMs MK-7622, PF-06764427, and PF-06827443. Moreover, VU0486846 shows no interaction with antagonist binding at the orthosteric acetylcholine (ACh) site (e.g., neither bitopic nor displaying negative cooperativity with [H]-NMS binding at the orthosteric site), no seizure liability at high brain exposures, and no cholinergic AEs. However, as opposed to ago-PAMs, VU0486846 produces robust efficacy in the novel object recognition model of cognitive function. Importantly, we show for the first time that an M PAM can reverse the cognitive deficits induced by atypical antipsychotics, such as risperidone. These findings further strengthen the argument that compounds with modest in vitro M PAM activity (EC > 100 nM) and pure-PAM activity in native tissues display robust procognitive efficacy without AEs mediated by excessive activation of M. Overall, the combination of compound assessment with recombinant in vitro assays (mindful of receptor reserve), native tissue systems (PFC), and phenotypic screens (behavioral convulsions) is essential to fully understand and evaluate lead compounds and enhance success in clinical development.
Positive allosteric modulators (PAMs) of the M subtype of muscarinic acetylcholine receptor have attracted intense interest as an exciting new approach for improving the cognitive deficits in schizophrenia and Alzheimer's disease. Recent evidence suggests that the presence of intrinsic agonist activity of some M PAMs may reduce efficacy and contribute to adverse effect liability. However, the M PAM PF-06827443 was reported to have only weak agonist activity at human M receptors but produced M-dependent adverse effects. We now report that PF-06827443 is an allosteric agonist in cell lines expressing rat, dog, and human M and use of inducible cell lines shows that agonist activity of PF-06827443 is dependent on receptor reserve. Furthermore, PF-06827443 is an agonist in native tissue preparations and induces behavioral convulsions in mice similar to other ago-PAMs. These findings suggest that PF-06827443 is a robust ago-PAM, independent of species, in cell lines and native systems.
Highly selective positive allosteric modulators (PAMs) of the M subtype of muscarinic acetylcholine receptor have emerged as an exciting new approach for improving cognitive function in patients suffering from Alzheimer's disease and schizophrenia. However, excessive activation of M is known to induce seizure activity and have actions in the prefrontal cortex (PFC) that could impair cognitive function. We now report a series of pharmacological, electrophysiological, and behavioral studies in which we find that recently reported M PAMs, PF-06764427 and MK-7622, have robust agonist activity in cell lines and agonist effects in the mouse PFC, and have the potential to overactivate the M receptor and disrupt PFC function. In contrast, structurally distinct M PAMs (VU0453595 and VU0550164) are devoid of agonist activity in cell lines and maintain activity dependence of M activation in the PFC. Consistent with the previously reported effect of PF-06764427, the ago-PAM MK-7622 induces severe behavioral convulsions in mice. In contrast, VU0453595 does not induce behavioral convulsions at doses well above those required for maximal efficacy in enhancing cognitive function. Furthermore, in contrast to the robust efficacy of VU0453595, the ago-PAM MK-7622 failed to improve novel object recognition, a rodent assay of cognitive function. These findings suggest that in vivo cognition-enhancing efficacy of M PAMs can be observed with PAMs lacking intrinsic agonist activity and that intrinsic agonist activity of M PAMs may contribute to adverse effects and reduced efficacy in improving cognitive function.
Although inhibition of phosphoinositide 3-kinase (PI3K) is an emerging strategy in cancer therapy, we and others have reported that this action can also contribute to drug-induced QT prolongation and arrhythmias by increasing cardiac late sodium current (I). Previous studies in mice implicate the PI3K- isoform in arrhythmia susceptibility. Here, we have determined the effects of new anticancer drugs targeting specific PI3K isoforms on I and action potentials (APs) in mouse cardiomyocytes and Chinese hamster ovary cells (CHO). Chronic exposure (10-100 nM; 5-48 hours) to PI3K--specific subunit inhibitors BYL710 (alpelisib) and A66 and a pan-PI3K inhibitor (BKM120) increased I in -transfected CHO cells and mouse cardiomyocytes. The specific inhibitors (10-100 nM for 5 hours) markedly prolonged APs and generated triggered activity in mouse cardiomyocytes (9/12) but not in controls (0/6), and BKM120 caused similar effects (3/6). The inclusion of water-soluble PIP3, a downstream effector of the PI3K signaling pathway, in the pipette solution reversed these arrhythmogenic effects. By contrast, inhibition of PI3K-, -, and - isoforms did not alter I or APs. We conclude that inhibition of cardiac PI3K- is arrhythmogenic by increasing I and this effect is not seen with inhibition of other PI3K isoforms. These results highlight a mechanism underlying potential cardiotoxicity of PI3K- inhibitors.
Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
C metabolic flux analysis (MFA) provides a rigorous approach to quantify intracellular metabolism of industrial cell lines. In this study, C MFA was used to characterize the metabolic response of Chinese hamster ovary (CHO) cells to a novel medium variant designed to reduce ammonia production. Ammonia inhibits growth and viability of CHO cell cultures, alters glycosylation of recombinant proteins, and enhances product degradation. Ammonia production was reduced by manipulating the amino acid composition of the culture medium; specifically, glutamine, glutamate, asparagine, aspartate, and serine levels were adjusted. Parallel C flux analysis experiments determined that, while ammonia production decreased by roughly 40%, CHO cell metabolic phenotype, growth, viability, and monoclonal antibody (mAb) titer were not significantly altered by the changes in media composition. This study illustrates how C flux analysis can be applied to assess the metabolic effects of media manipulations on mammalian cell cultures. The analysis revealed that adjusting the amino acid composition of CHO cell culture media can effectively reduce ammonia production while preserving fluxes throughout central carbon metabolism.
© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Poly(lactic-co-glycolic acid) (PLGA) is widely used as a vehicle for delivery of pharmaceutically relevant payloads. PLGA is readily fabricated as a nano- or microparticle (MP) matrix to load both hydrophobic and hydrophilic small molecular drugs as well as biomacromolecules such as nucleic acids and proteins. However, targeting such payloads to the cell cytosol is often limited by MP entrapment and degradation within acidic endolysosomes. Poly(propylacrylic acid) (PPAA) is a polyelectrolyte polymer with the membrane disruptive capability triggered at low pH. PPAA has been previously formulated in various carrier configurations to enable cytosolic payload delivery, but requires sophisticated carrier design. Taking advantage of PPAA functionality, we have incorporated PPAA into PLGA MPs as a simple polymer mixture to enhance cytosolic delivery of PLGA-encapsulated payloads. Rhodamine loaded PLGA and PPAA/PLGA blend MPs were prepared by a modified nanoprecipitation method. Incorporation of PPAA into PLGA MPs had little to no effect on the size, shape, or loading efficiency, and evidenced no toxicity in Chinese hamster ovary epithelial cells. Notably, incorporation of PPAA into PLGA MPs enabled pH-dependent membrane disruption in a hemolysis assay, and a three-fold increased endosomal escape and cytosolic delivery in dendritic cells after 2 h of MP uptake. These results demonstrate that a simple PLGA/PPAA polymer blend is readily fabricated into composite MPs, enabling cytosolic delivery of an encapsulated payload. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1022-1033, 2018.
© 2017 Wiley Periodicals, Inc.