Hepatitis C virus modulation of lipid and autophagy pathways

Hepatitis C virus [HCV] infects 170 million people worldwide. We investigated interactions between HCV proteins and cellular proteins involved in autophagy and lipid metabolism. We sought to develop an infection model using patient derived human serum containing HCV and human hepatocytes, Huh7 cells. Using the model, we have shown intracellular expression of incoming HCV RNA (5′ UTR region and region spanning the E1/E2 glycoproteins), expression of the HCV proteins, core and NS5B, and a cellular response to HCV infection. These data suggests this model can be used to analyse the early stage of HCV infection. HCV utilises the autophagy pathway to both establish infection and to complete its life cycle. We investigated HCV interaction with the early stage autophagy protein ATG5. We found that although ATG5 mRNA is unchanged in HCV infected cells, protein expression of ATG5 is significantly upregulated. These data indicated HCV controls the post-transcriptional regulation of ATG5. We used the upstream open reading frame (uORF) and the 5′ UTR region of ATG5 to examine the post-transcriptional regulation. Our data suggest HCV RNA replication either directly or indirectly causes post-transcriptional regulation of the early autophagy protein, ATG5 in a 5′ UTR and uORF independent manner. HCV infection leads to an increase in SREBP controlled genes e.g. HMG-CoA Reductase, cholesterol, LDL and fatty acid synthesis. We hypothesised that HCV infection causes the activation of SREBP pathway by interacting directly or indirectly with proteins involved in the initiation of the pathway. We sought to determine if HCV interacts with SCAP or INSIG. We confirmed a change in LD distribution and HMG-CoA reductase activity as a result of HCV RNA replication. Significantly, we show SCAP protein expression was also altered during HCV RNA replication and HCV core protein possibly interacts with SCAP.

Implication de la convertase NARC-1 / PCSK9 au cours de la différenciation neuroectodermale

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

STOPPFrail (Screening Tool of Older Persons Prescriptions in Frail Adults with Limited Life Expectancy): Consensus Validation

Background: To validate STOPPFrail, a list of explicit criteria for potentially inappropriate medications (PIMs) in frailer older adults with limited life expectancy. A Delphi consensus survey of an expert panel (n = 17) comprising specialists in geriatric medicine, clinical pharmacology, palliative care, psychiatry of old age, clinical pharmacy and general practice.
Methods: STOPPFrail criteria was initially created by the authors based on clinical
experience and appraisal of the available literature. Criteria were organised according to physiological system. Each criterion was accompanied by an explanation. Panellists ranked their agreement with each criterion on a 5-point Likert scale and invited to provide written feedback. Criteria with a median Likert response of 4/5 (agree/strongly agree) and a 25th centile of ≥4 were included in the final criteria.
Results: Three Delphi rounds were required. All panellists completed all rounds. Thirty criteria were proposed for inclusion; 26 were accepted. No new criteria were added. The first two criteria suggest deprescribing medications with no indication or where compliance is poor. The remaining 24 criteria include lipid-lowering therapies, alpha-blockers for hypertension, anti-platelets, neuroleptics, proton pump inhibitors, H-2 receptor antagonists, anti-spasmodics, theophylline, leukotriene antagonists, calcium supplements, bone anti-resorptive therapy, selective oestrogen receptor modulators, non-steroidal antiinflammatories, corticosteroids, 5-alpha reductase inhibitors, alpha-1 selective blockers, muscarinic antagonists, oral diabetic agents, ACE-inhibitors, angiotensin receptor blockers, systemic oestrogens, multivitamins, nutritional supplements and prophylactic antibiotics. Anticoagulants and anti-depressants were excluded. Despite incorporation of panellists’ suggestions, memantine and acetyl-cholinesterase inhibitors remained inconclusive.
Conclusion: STOPPFrail comprises 26 criteria, which have been judged by broad consensus, to be potentially inappropriate in frailer older patients with limited life expectancy. STOPPFrail may assist in deprescribing medications in these patients.

Impaired reverse cholesterol transport and hepatic steatosis contribute to pathogenesis of high fat dietinduced hyperlipidemia in murine models

Purpose: To investigate the pathogenesis of high fat diet (HFD)-induced hyperlipidemia (HLP) in mice, rats and hamsters and to comparatively evaluate their sensitivity to HFD. Methods: Mice, rats and hamsters were fed with high-fat diet formulation (HFD, n = 8) or a control diet (control, n = 8) for 4 weeks. Changes in body weight, relative liver weight, serum lipid profile, expressions of hepatic marker gene of lipid metabolism and liver morphology were observed in three hyperlipidemic models. Results: Elevated total cholesterol (TC), triglyceride, low density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL-C) levels and body weight were observed in all hyperlipidemic animals (p < 0.05), while hepatic steatosis was manifested in rat and hamster HLP models, and increased hepatic TC level was only seen (p < 0.05) in hamster HLP model. Suppression of HMG-CoA reductase and up-regulation of lipoproteinlipase were observed in all HFD groups. Hepatic gene expression of LDLR, CYP7A1, LCAT, SR-B1, and ApoA I, which are a response to reverse cholesterol transport (RCT), were inhibited by HFD in the three models. Among these models, simultaneous suppression of HMG-CR, LCAT, LDLR and SR-BI and elevated LPL were features of the hamster model. Conclusion: As the results show, impaired RCT and excessive fat accumulation are major contributors to pathogenesis of HFD-induced murine HLP. Thus, the hamster model is more appropriate for hyperlipidemia research.

Design, synthesis, and biological and crystallographic evaluation of novel inhibitors of Plasmodium falciparum enoyl-ACP-reductase (PfFabI).

Malaria, a disease of worldwide significance, is responsible for over one million deaths annually. The liver-stage of Plasmodium's life cycle is the first, obligatory, but clinically silent step in malaria infection. The P. falciparum type II fatty acid biosynthesis pathway (PfFAS-II) has been found to be essential for complete liver-stage development and has been regarded as a potential antimalarial target for the development of drugs for malaria prophylaxis and liver-stage eradication. In this paper, new coumarin-based triclosan analogues are reported and their biological profile is explored in terms of inhibitory potency against enzymes of the PfFAS-II pathway. Among the tested compounds, 7 and 8 showed the highest inhibitory potency against Pf enoyl-ACP-reductase (PfFabI), followed by 15 and 3. Finally, we determined the crystal structures of compounds 7 and 11 in complex with PfFabI to identify their mode of binding and to confirm outcomes of docking simulations.

Three-Dimensional Quantitative Structure-Activity Relationship Study of Antitumor 2-Formylpyridine Thiosemicarbazones Derivatives as Inhibitors of Ribonucleotide Reductase

In order to extend previous SAR and QSAR studies, 3D-QSAR analysis has been performed using CoMFA and CoMSIA approaches applied to a set of 39 alpha-(N)-heterocyclic carboxaldehydes thiosemicarbazones with their inhibitory activity values (IC(50)) evaluated against ribonucleotide reductase (RNR) of H.Ep.-2 cells (human epidermoid carcinoma), taken from selected literature. Both rigid and field alignment methods, taking the unsubstituted 2-formylpyridine thiosemicarbazone in its syn conformation as template, have been used to generate multiple predictive CoMFA and CoMSIA models derived from training sets and validated with the corresponding test sets. Acceptable predictive correlation coefficients (Q(cv)(2) from 0.360 to 0.609 for CoMFA and Q(cv)(2) from 0.394 to 0.580 for CoMSIA models) with high fitted correlation coefficients (r` from 0.881 to 0.981 for CoMFA and r(2) from 0.938 to 0.993 for CoMSIA models) and low standard errors (s from 0.135 to 0.383 for CoMFA and s from 0.098 to 0.240 for CoMSIA models) were obtained. More precise CoMFA and CoMSIA models have been derived considering the subset of thiosemicarbazones (TSC) substituted only at 5-position of the pyridine ring (n=22). Reasonable predictive correlation coefficients (Q(cv)(2) from 0.486 to 0.683 for CoMFA and Q(cv)(2) from 0.565 to 0.791 for CoMSIA models) with high fitted correlation coefficients (r(2) from 0.896 to 0.997 for CoMFA and r(2) from 0.991 to 0.998 for CoMSIA models) and very low standard errors (s from 0.040 to 0.179 for CoMFA and s from 0.029 to 0.068 for CoMSIA models) were obtained. The stability of each CoMFA and CoMSIA models was further assessed by performing bootstrapping analysis. For the two sets the generated CoMSIA models showed, in general, better statistics than the corresponding CoMFA models. The analysis of CoMFA and CoMSIA contour maps suggest that a hydrogen bond acceptor near the nitrogen of the pyridine ring can enhance inhibitory activity values. This observation agrees with literature data, which suggests that the nitrogen pyridine lone pairs can complex with the iron ion leading to species that inhibits RNR. The derived CoMFA and CoMSIA models contribute to understand the structural features of this class of TSC as antitumor agents in terms of steric, electrostatic, hydrophobic and hydrogen bond donor and hydrogen bond acceptor fields as well as to the rational design of this key enzyme inhibitors.

Novel aryl beta-aminocarbonyl derivatives as inhibitors of Trypanosoma cruzi trypanothione reductase: binding mode revised by docking and GRIND2-based 3D-QSAR procedures

Trypanothione reductase has long been investigated as a promising target for chemotherapeutic intervention in Chagas disease, since it is an enzyme of a unique metabolic pathway that is exclusively present in the pathogen but not in the human host, which has the analog Glutathione reductase. In spite of the present data-set includes a small number of compounds, a combined use of flexible docking, pharmacophore perception, ligand binding site prediction, and Grid-Independent Descriptors GRIND2-based 3D-Quantitative Structure-Activity Relationships (QSAR) procedures allowed us to rationalize the different biological activities of a series of 11 aryl beta-aminocarbonyl derivatives, which are inhibitors of Trypanosoma cruzi trypanothione reductase (TcTR). Three QSAR models were built and validated using different alignments, which are based on docking with the TcTR crystal structure, pharmacophore, and molecular interaction fields. The high statistical significance of the models thus obtained assures the robustness of this second generation of GRIND descriptors here used, which were able to detect the most important residues of such enzyme for binding the aryl beta-aminocarbonyl derivatives, besides to rationalize distances among them. Finally, a revised binding mode has been proposed for our inhibitors and independently supported by the different methodologies here used, allowing further optimization of the lead compounds with such combined structure- and ligand-based approaches in the fight against the Chagas disease.

Inhibitors of Trypanosoma brucei trypanothione reductase: comparative molecular field analysis modeling and structural basis for selective inhibition

Background: Sleeping sickness is a major cause of death in Africa. Since no secure treatment is available, the development of novel therapeutic agents is urgent. In this context, the enzyme trypanothione reductase (TR) is a prominent molecular target that has been investigated in drug design for sleeping sickness. Results: In this study, comparative molecular field analysis models were generated for a series of Trypanosoma brucei TR inhibitors. Statistically significant results were obtained and the models were applied to predict the activity of external test sets, with good correlation between predicted and experimental results. We have also investigated the structural requirements for the selective inhibition of the parasite's enzyme over the human glutathione reductase. Conclusion: The quantitative structure-activity relationship models provided valuable information regarding the essential molecular requirements for the inhibitory activity upon the target protein, providing important insights into the design of more potent and selective TR inhibitors.

Theoretical investigation of the [1,2]-sigmatropic hydrogen migration in the mechanism of oxidation of 2-aminobenzoyl-CoA by 2-aminobenzoyl-CoA monooxygenase/reductase

The flavin hydroperoxide at the active site of the mixed-function oxidase 2-aminobenzoyl-CoA monooxygenase/reductase (Azoarcus evansii) transfers an oxygen to the 5-position of the 2-aminobenzoyl-CoA substrate to provide the alkoxide intermediate II−. Hydrogen migration from C5 to C6 follows this monooxygenation. The nature of the monooxygenation intermediate and plausible competing reactions leading to hydrogen migration have been considered. Ab initio molecular orbital theory has been used to calculate structures and electron distributions in intermediate and transition state structures. Electrostatic potential surface calculations establish that the transition state and product, associated with the C5 to C6 hydrogen transfer, are stabilized by electron distribution to the benzoyl-CoA thioester carbonyl oxygen. This is not so for the transition state and product associated with hydrogen transfer from C5 to C4. The activation energy for the 5,6-shift is 2.5 kcal/mol lower than that for the 5,4-shift. In addition, the product of the hydrogen 5,6-shift is more stable than is the product of the hydrogen 5,4-shift, by ≈6 kcal/mol. These results explain why only the shift of hydrogen from C5 to C6 is observed experimentally. Oxygen transfer and hydrogen migration almost coincide in the gas phase (activation energy of ≈0.6 kcal/mol, equivalent to a single bond vibration). Enzymatic formation of alkoxide II− requires its stabilization; thus, the rate constant for its breakdown would be slower than in the gas phase.

An isoleucine/leucine residue in the carboxyltransferase domain of acetyl-CoA carboxylase is critical for interaction with aryloxyphenoxypropionate and cyclohexanedione inhibitors

cDNA fragments encoding the carboxyltransferase domain of the multidomain plastid acetyl-CoA carboxylase (ACCase) from herbicide-resistant maize and from herbicide-sensitive and herbicide-resistant Lolium rigidum were cloned and sequenced. A Leu residue was found in ACCases from herbicide-resistant plants at a position occupied by Ile in all ACCases from sensitive grasses studied so far. Leu is present at the equivalent position in herbicide-resistant ACCases from other eukaryotes. Chimeric ACCases containing a 1000-aa fragment of two ACCase isozymes found in a herbicide-resistant maize were expressed in a yeast ACC1 null mutant to test herbicide sensitivity of the enzyme in vivo and in vitro. One of the enzymes was resistant/tolerant, and one was sensitive to haloxyfop and sethoxydim, rendering the gene-replacement yeast strains resistant and sensitive to these compounds, respectively. The sensitive enzyme has an Ile residue, and the resistant one has a Leu residue at the putative herbicide-binding site. Additionally, a single Ile to Leu replacement at an equivalent position changes the wheat plastid ACCase from sensitive to resistant. The effect of the opposite substitution, Leu to Ile, makes Toxoplasma gondii apicoplast ACCase resistant to haloxyfop and clodinafop. In this case, inhibition of the carboxyltransferase activity of ACCase (second half-reaction) of a large fragment of the Toxoplasma enzyme expressed in Escherichia coli was tested. The critical amino acid residue is located close to a highly conserved motif of the carboxyltransferase domain, which is probably a part of the enzyme active site, providing the basis for the activity of fop and dim herbicides.

Non-classical inhibitors of dihydrofolate reductase

This thesis comprises two main objectives. The first objective involved the stereochemical studies of chiral 4,6-diamino-1-aryl-1,2-dihydro-s-triazines and an investigation on how the different conformations of these stereoisomers may affect their binding affinity to the enzyme dihydrofolate reductase (DHFR). The ortho-substituted 1-aryl-1,2-dihydro-s-triazines were synthesised by the three component method. An ortho-substitution at the C6' position was observed when meta-azidocycloguanil was decomposed in acid. The ortho-substituent restricts free rotation and this gives rise to atropisomerism. Ortho-substituted 4,6-diamino-1-aryl-2-ethyl-1,2-dihydro-2-methyl-s-triazine contains two elements of chirality and therefore exists as four stereoisomers: (S,aR), (R,aS), (R,aR) and (S,aS). The energy barriers to rotation of these compounds were calculated by a semi-empirical molecular orbital program called MOPAC and they were found to be in excess of 23 kcal/mol. The diastereoisomers were resolved and enriched by C18 reversed phase h.p.l.c. Nuclear overhauser effect experiments revealed that (S,aR) and (R,aS) were the more stable pair of stereoisomers and therefore existed as the major component. The minor diastereoisomers showed greater binding affinity for the rat liver DHFR in in vitro assay. The second objective entailed the investigation into the possibility of retaining DHFR inhibitory activity by replacing the classical diamino heterocyclic moiety with an amidinyl group. 4-Benzylamino-3-nitro-N,N-dimethyl-phenylamidine was synthesised in two steps. One of the two phenylamidines indicated weak inhibition against the rat liver DHFR. This weak activity may be due to the failure of the inhibitor molecule to form strong hydrogen bonds with residue Glu-30 at the active site of the enzyme.

Design and synthesis of inhibitors of dihydrofolate reductase

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT