Biochemistry and Pharmacology - Research Publications
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ItemExtinction of a cocaine-taking context that protects against drug-primed reinstatement is dependent on the metabotropic glutamate 5 receptor(WILEY, 2015-05)We investigated the effects of extinguishing action-reward versus context-reward associations on drug-primed reinstatement, and the potential role of the metabotropic glutamate 5 receptor (mGlu5) in these different types of extinction in rats that self-administer cocaine. We observed that daily context extinction (non-reinforced exposures to the cocaine-taking context with retracted levers) was just as effective as daily lever extinction in reducing cocaine-primed reinstatement compared with passive abstinence. Additionally, systemic injections of the mGlu5 negative allosteric modulator MTEP (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine) following each extinction session significantly impaired the ability of context extinction to reduce cocaine-primed reinstatement, without affecting reinstatement after lever extinction or passive abstinence.
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ItemP2X7 Receptor-mediated Scavenger Activity of Mononuclear Phagocytes toward Non-opsonized Particles and Apoptotic Cells Is Inhibited by Serum Glycoproteins but Remains Active in Cerebrospinal Fluid(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012-05-18)Rapid phagocytosis of non-opsonized particles including apoptotic cells is an important process that involves direct recognition of the target by multiple scavenger receptors including P2X7 on the phagocyte surface. Using a real-time phagocytosis assay, we studied the effect of serum proteins on this phagocytic process. Inclusion of 1-5% serum completely abolished phagocytosis of non-opsonized YG beads by human monocytes. Inhibition was reversed by pretreatment of serum with 1-10 mM tetraethylenepentamine, a copper/zinc chelator. Inhibitory proteins from the serum were determined as negatively charged glycoproteins (pI < 6) with molecular masses between 100 and 300 kDa. A glycoprotein-rich inhibitory fraction of serum not only abolished YG bead uptake but also inhibited phagocytosis of apoptotic lymphocytes or neuronal cells by human monocyte-derived macrophages. Three copper- and/or zinc-containing serum glycoproteins, ceruloplasmin, serum amyloid P-component, and amyloid precursor protein, were identified, and the purified proteins were shown to inhibit the phagocytosis of beads by monocytes as well as phagocytosis of apoptotic neuronal cells by macrophages. Human adult cerebrospinal fluid, which contains very little glycoprotein, had no inhibitory effect on phagocytosis of either beads or apoptotic cells. These data suggest for the first time that metal-interacting glycoproteins present within serum are able to inhibit the scavenger activity of mononuclear phagocytes toward insoluble debris and apoptotic cells.
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ItemIn silico prediction of antimalarial drug target candidates(ELSEVIER SCI LTD, 2012-12)The need for new antimalarials is persistent due to the emergence of drug resistant parasites. Here we aim to identify new drug targets in Plasmodium falciparum by phylogenomics among the Plasmodium spp. and comparative genomics to Homo sapiens. The proposed target discovery pipeline is largely independent of experimental data and based on the assumption that P. falciparum proteins are likely to be essential if (i) there are no similar proteins in the same proteome and (ii) they are highly conserved across the malaria parasites of mammals. This hypothesis was tested using sequenced Saccharomycetaceae species as a touchstone. Consecutive filters narrowed down the potential target space of P. falciparum to proteins that are likely to be essential, matchless in the human proteome, expressed in the blood stages of the parasite, and amenable to small molecule inhibition. The final set of 40 candidate drug targets was significantly enriched in essential proteins and comprised proven targets (e.g. dihydropteroate synthetase or enzymes of the non-mevalonate pathway), targets currently under investigation (e.g. calcium-dependent protein kinases), and new candidates of potential interest such as phosphomannose isomerase, phosphoenolpyruvate carboxylase, signaling components, and transporters. The targets were prioritized based on druggability indices and on the availability of in vitro assays. Potential inhibitors were inferred from similarity to known targets of other disease systems. The identified candidates from P. falciparum provide insight into biochemical peculiarities and vulnerable points of the malaria parasite and might serve as starting points for rational drug discovery.
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ItemBiosynthesis, Localization, and Macromolecular Arrangement of the Plasmodium falciparum Translocon of Exported Proteins (PTEX)(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012-03-09)To survive within its host erythrocyte, Plasmodium falciparum must export hundreds of proteins across both its parasite plasma membrane and surrounding parasitophorous vacuole membrane, most of which are likely to use a protein complex known as PTEX (Plasmodium translocon of exported proteins). PTEX is a putative protein trafficking machinery responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex, and in this study, three of the major stoichiometric components are investigated including HSP101 (a AAA(+) ATPase), a protein of no known function termed PTEX150, and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont stage (PTEX150 and HSP101) or even earlier in the life cycle (EXP2), and before invasion these components reside within the dense granules of invasive merozoites. From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230 kDa that includes an ∼600-kDa apparently homo-oligomeric complex of EXP2 that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components associate as EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerizes and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.
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ItemStimulating the Release of Exosomes Increases the Intercellular Transfer of Prions(ELSEVIER, 2016-03-04)Exosomes are small extracellular vesicles released by cells and play important roles in intercellular communication and pathogen transfer. Exosomes have been implicated in several neurodegenerative diseases, including prion disease and Alzheimer disease. Prion disease arises upon misfolding of the normal cellular prion protein, PrP(C), into the disease-associated isoform, PrP(Sc). The disease has a unique transmissible etiology, and exosomes represent a novel and efficient method for prion transmission. The precise mechanism by which prions are transmitted from cell to cell remains to be fully elucidated, although three hypotheses have been proposed: direct cell-cell contact, tunneling nanotubes, and exosomes. Given the reported presence of exosomes in biological fluids and in the lipid and nucleic acid contents of exosomes, these vesicles represent an ideal mechanism for encapsulating prions and potential cofactors to facilitate prion transmission. This study investigates the relationship between exosome release and intercellular prion dissemination. Stimulation of exosome release through treatment with an ionophore, monensin, revealed a corresponding increase in intercellular transfer of prion infectivity. Conversely, inhibition of exosome release using GW4869 to target the neutral sphingomyelinase pathway induced a decrease in intercellular prion transmission. Further examination of the effect of monensin on PrP conversion revealed that monensin also alters the conformational stability of PrP(C), leading to increased generation of proteinase K-resistant prion protein. The findings presented here provide support for a positive relationship between exosome release and intercellular transfer of prion infectivity, highlighting an integral role for exosomes in facilitating the unique transmissible nature of prions.
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ItemNrdR Transcription Regulation: Global Proteome Analysis and Its Role in Escherichia coli Viability and Virulence(PUBLIC LIBRARY SCIENCE, 2016-06-08)Bacterial ribonucleotide reductases (RNRs) play an important role in the synthesis of dNTPs and their expression is regulated by the transcription factors, NrdR and Fur. Recent transcriptomic studies using deletion mutants have indicated a role for NrdR in bacterial chemotaxis and in the maintenance of topoisomerase levels. However, NrdR deletion alone has no effect on bacterial growth or virulence in infected flies or in human blood cells. Furthermore, transcriptomic studies are limited to the deletion strain alone, and so are inadequate for drawing biological implications when the NrdR repressor is active or abundant. Therefore, further examination is warranted of changes in the cellular proteome in response to both NrdR overexpression, as well as deletion, to better understand its functional relevance as a bacterial transcription repressor. Here, we profile bacterial fate under conditions of overexpression and deletion of NrdR in E. coli. Biochemical assays show auxiliary zinc enhances the DNA binding activity of NrdR. We also demonstrate at the physiological level that increased nrdR expression causes a significant reduction in bacterial growth and fitness even at normal temperatures, and causes lethality at elevated temperatures. Corroborating these direct effects, global proteome analysis following NrdR overexpression showed a significant decrease in global protein expression. In parallel, studies on complementary expression of downregulated essential genes polA, eno and thiL showed partial rescue of the fitness defect caused by NrdR overexpression. Deletion of downregulated non-essential genes ygfK and trxA upon NrdR overexpression resulted in diminished bacterial growth and fitness suggesting an additional role for NrdR in regulating other genes. Moreover, in comparison with NrdR deletion, E. coli cells overexpressing NrdR showed significantly diminished adherence to human epithelial cells, reflecting decreased bacterial virulence. These results suggest that elevated expression of NrdR could be a suitable means to retard bacterial growth and virulence, as its elevated expression reduces bacterial fitness and impairs host cell adhesion.
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ItemStructure and inhibition of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus(WILEY, 2016-12)N-Acetylneuraminate lyase is the first committed enzyme in the degradation of sialic acid by bacterial pathogens. In this study, we analyzed the kinetic parameters of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus (MRSA). We determined that the enzyme has a relatively high KM of 3.2 mm, suggesting that flux through the catabolic pathway is likely to be controlled by this enzyme. Our data indicate that sialic acid alditol, a known inhibitor of N-acetylneuraminate lyase enzymes, is a stronger inhibitor of MRSA N-acetylneuraminate lyase than of Clostridium perfringens N-acetylneuraminate lyase. Our analysis of the crystal structure of ligand-free and 2R-sialic acid alditol-bound MRSA N-acetylneuraminate lyase suggests that subtle dynamic differences in solution and/or altered binding interactions within the active site may account for species-specific inhibition.
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ItemNovel mechanism of modulation at a ligand-gated ion channel; action of 5-Cl-indole at the 5-HT3A receptor(WILEY-BLACKWELL, 2016-12)BACKGROUND AND PURPOSE: The 5-HT3 receptor is a prototypical member of the Cys-loop ligand-gated ion channel (LGIC) superfamily and an established therapeutic target. In addition to activation via the orthosteric site, receptor function can be modulated by allosteric ligands. We have investigated the pharmacological action of Cl-indole upon the 5-HT3 A receptor and identified that this positive allosteric modulator possesses a novel mechanism of action for LGICs. EXPERIMENTAL APPROACH: The impact of Cl-indole upon the 5-HT3 receptor was assessed using single cell electrophysiological recordings and [3 H]-granisetron binding in HEK293 cells stably expressing the 5-HT3 receptor. KEY RESULTS: Cl-indole failed to evoke 5-HT3 A receptor-mediated responses (up to 30 μM) or display affinity for the [3 H]-granisetron binding site. However, in the presence of Cl-indole, termination of 5-HT application revealed tail currents mediated via the 5-HT3 A receptor that were independent of the preceding 5-HT concentration but were antagonized by the 5-HT3 receptor antagonist, ondansetron. These tail currents were absent in the 5-HT3 AB receptor. Furthermore, the presence of 5-HT revealed a concentration-dependent increase in the affinity of Cl-indole for the orthosteric binding site of the human 5-HT3 A receptor. CONCLUSIONS AND IMPLICATIONS: Cl-indole acts as both an orthosteric agonist and an allosteric modulator, but the presence of an orthosteric agonist (e.g. 5-HT) is a prerequisite to reveal both actions. Precedent for ago-allosteric action is available, yet the essential additional presence of an orthosteric agonist is now reported for the first time. This widening of the pharmacological mechanisms to modulate LGICs may offer further therapeutic opportunities.
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ItemType-1 Interferons Contribute to the Neuroinflammatory Response and Disease Progression of the MPTP Mouse Model of Parkinson's Disease(WILEY, 2016-09)Type-1 interferons (IFNs) are pleiotropic cytokines with a critical role in the initiation and regulation of the pro-inflammatory response. However, the contribution of the type-1 IFNs to CNS disorders, specifically chronic neuropathologies such as Parkinson's disease is still unknown. Here, we report increased type-1 IFN signaling in both post mortem human Parkinson's disease samples and in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) mouse model. In response to MPTP, mice lacking the type-1 IFN receptor (IFNAR1(-/-) ) displayed decreased type-1 IFN signaling, an attenuated pro-inflammatory response and reduced loss of dopaminergic neurons. The neuroprotective potential of targeting the type-1 IFN pathway was confirmed by reduced neuroinflammation and DA cell death in mice treated with a blocking monoclonal IFNAR1 (MAR-1) antibody. The MPTP/MAR-1 treated mice also displayed increased striatal dopamine levels and improved behavioural outcomes compared to their MPTP/IgG controls. These data, implicate for the first time, a deleterious role for the type-1 IFNs as key modulators of the early neuroinflammatory response and therefore the neuronal cell death in Parkinson's disease. GLIA 2016;64:1590-1604.
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ItemThe inhibitor of semicarbazide-sensitive amine oxidase, PXS-4728A, ameliorates key features of chronic obstructive pulmonary disease in a mouse model(WILEY, 2016-11)BACKGROUND AND PURPOSE: Chronic obstructive pulmonary disease (COPD) is a major cause of illness and death, often induced by cigarette smoking (CS). It is characterized by pulmonary inflammation and fibrosis that impairs lung function. Existing treatments aim to control symptoms but have low efficacy, and there are no broadly effective treatments. A new potential target is the ectoenzyme, semicarbazide-sensitive mono-amine oxidase (SSAO; also known as vascular adhesion protein-1). SSAO is elevated in smokers' serum and is a pro-inflammatory enzyme facilitating adhesion and transmigration of leukocytes from the vasculature to sites of inflammation. EXPERIMENTAL APPROACH: PXS-4728A was developed as a low MW inhibitor of SSAO. A model of COPD induced by CS in mice reproduces key aspects of human COPD, including chronic airway inflammation, fibrosis and impaired lung function. This model was used to assess suppression of SSAO activity and amelioration of inflammation and other characteristic features of COPD. KEY RESULTS: Treatment with PXS-4728A completely inhibited lung and systemic SSAO activity induced by acute and chronic CS-exposure. Daily oral treatment inhibited airway inflammation (immune cell influx and inflammatory factors) induced by acute CS-exposure. Therapeutic treatment during chronic CS-exposure, when the key features of experimental COPD develop and progress, substantially suppressed inflammatory cell influx and fibrosis in the airways and improved lung function. CONCLUSIONS AND IMPLICATIONS: Treatment with a low MW inhibitor of SSAO, PXS-4728A, suppressed airway inflammation and fibrosis and improved lung function in experimental COPD, demonstrating the therapeutic potential of PXS-4728A for this debilitating disease.