DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors.

Viral vectors based on adeno-associated virus (AAV) preferentially transduce cells in S phase of the cell cycle. We recently found that DNA-damaging agents increased the transduction of nondividing cells. However, the optimal concentrations were toxic to cells. Here we show that the transduction of normal human fibroblasts by AAV vectors is increased by prior exposure to DNA synthesis inhibitors, such as aphidicolin or hydroxyurea, and topoisomerase inhibitors, such as etoposide or camptothecin. Transduction efficiencies could be increased > 300-fold in stationary cultures at concentrations that did not affect cell viability or proliferative potential. Both S-phase and non-S-phase cells were affected, suggesting that cellular functions other than replicative DNA synthesis may be involved. Applying these methods to gene transfer protocols should improve prospects for gene therapy by AAV vectors.

Suppression of heat-induced hsp70 expression by the 70-kDa subunit of the human Ku autoantigen.

Expression of the 70-kDa polypeptide of human Ku autoantigen in rat cells is shown to suppress specifically the induction of hsp70 upon heat shock. Thermal induction of other heat shock proteins is not significantly affected, nor is the state of phosphorylation or the DNA-binding ability of the heat shock transcription factor HSF1. These findings support a model in which hsp70 gene expression is controlled by a second regulatory factor in addition to the positive activator HSF1. The Ku autoantigen, or a protein closely related to it, is likely to be involved in the regulation of hsp70 expression.

Protein structure-based design of potent orally bioavailable, nonpeptide inhibitors of human immunodeficiency virus protease.

A class of potent nonpeptidic inhibitors of human immunodeficiency virus protease has been designed by using the three-dimensional structure of the enzyme as a guide. By employing iterative protein cocrystal structure analysis, design, and synthesis the binding affinity of the lead compound was incrementally improved by over four orders of magnitude. An inversion in inhibitor binding mode was observed crystallographically, providing information critical for subsequent design and highlighting the utility of structural feedback in inhibitor optimization. These inhibitors are selective for the viral protease enzyme, possess good antiviral activity, and are orally available in three species.

Ras farnesyltransferase inhibitors suppress the phenotype resulting from an activated ras mutation in Caenorhabditis elegans.

Attachment of Ras protein to the membrane, which requires farnesylation at its C terminus, is essential for its biological activity. A promising pharmacological approach of antagonizing oncogenic Ras activity is to develop inhibitors of farnesyltransferase. We use Caenorhabditis elegans vulval differentiation, which is controlled by a Ras-mediated signal transduction pathway, as a model system to test previously identified farnesyltransferase inhibitors. We show here that two farnesyltransferase inhibitors, manumycin and gliotoxin, suppress the Multivulva phenotype resulting from an activated let-60 ras mutation, but not the Multivulva phenotype resulting from mutations in the lin-1 gene or the lin-15 gene, which act downstream and upstream of let-60 ras, respectively, in the signaling pathway. These results are consistent with the idea that the suppression of the Multivulva phenotype of let-60 ras by the two inhibitors is specific for Ras protein and that the mutant Ras protein might be more sensitive than wild-type Ras to the farnesyltransferase inhibitors. This work suggests that C. elegans vulval development could be a simple and effective in vivo system for evaluation of farnesyltransferase inhibitors against Ras-activated tumors.

Design and synthesis of new multitarget inhibitors of dengue virus replication

Attraverso una combinazione di virtual screening/virtual library generation abbiamo identificato una nuova serie di inibitori della replicazione del virus Dengue, attivi sia su un target virale (NS5) sia su uno cellulare (Src chinasi)

The place of DPP-4 inhibitors in the treatment algorithm of diabetes type 2 : a systematic review of cost-effectiveness studies

Tese de mestrado, Epidemiologia, Universidade de Lisboa, Faculdade de Medicina, 2015

Implicating the mechanisms of ADP-ribosylation factor activation in the resistance of invasive breast cancer cells to EGFR tyrosine kinase inhibitors

ADP-ribosylation factor-1 (ARF1) est une petite GTPase principalement connue pour son rôle dans la formation de vésicules au niveau de l’appareil de Golgi. Récemment, dans des cellules de cancer du sein, nous avons démontré qu’ARF1 est aussi un médiateur important de la signalisation du récepteur du facteur de croissance épidermique (EGFR) contrôlant la prolifération, la migration et l'invasion cellulaire. Cependant, le mécanisme par lequel l’EGFR active la GTPase ainsi que le rôle de cette dernière dans la régulation de la fonction du récepteur demeure inconnue. Dans cette thèse, nous avions comme objectifs de définir le mécanisme d'activation de ARF1 dans les cellules de cancer du sein hautement invasif et démontrer que l’activation de cette isoforme de ARF joue un rôle essentiel dans la résistance de ces cellules aux inhibiteurs de l'EGFR. Nos études démontrent que les protéines d’adaptatrices Grb2 et p66Shc jouent un rôle important dans l'activation de ARF1. Alors que Grb2 favorise le recrutement d’ARF1 à l'EGFR ainsi que l'activation de cette petite GTPase, p66Shc inhibe le recrutement du complexe Grb2-ARF1 au récepteur et donc contribue à limiter l’activation d’ARF1. De plus, nous démontrons que ARF1 favorise la résistance aux inhibiteurs des tyrosines kinases dans les cellules de cancer du sein hautement invasif. En effet, une diminution de l’expression de ARF1 a augmenté la sensibilité descellules aux inhibiteurs de l'EGFR. Nous montrons également que de hauts niveaux de ARF1 contribuent à la résistance des cellules à ces médicaments en améliorant la survie et les signaux prolifératifs à travers ERK1/2, Src et AKT, tout en bloquant les voies apoptotiques (p38MAPK et JNK). Enfin, nous mettons en évidence le rôle de la protéine ARF1 dans l’apoptose en réponse aux traitements des inhibiteurs de l’EGFR. Nos résultats indiquent que la dépletion d’ARF1 promeut la mort cellulaire induite par gefitinib, en augmentant l'expression de facteurs pro-apoptotiques (p66shc, Bax), en altérant le potentiel de la membrane mitochondriale et la libération du cytochrome C. Ensemble, nos résultats délimitent un nouveau mécanisme d'activation de ARF1 dans les cellules du cancer du sein hautement invasif et impliquent l’activité d’ARF1 comme un médiateur important de la résistance aux inhibiteurs EGFR.

Proton pump inhibitors and the risk of fractures in older adults: a population-based retrospective cohort study

Thesis (Master's)--University of Washington, 2016-06

D-Tyrosine as a chiral precusor to potent inhibitors of human nonpancreatic secretory phospholipase A(2) (IIa) with antiinflammatory activity

Few reported inhibitors of secretory phospholipase A(2) enzymes inhibit the IIa human isoform (hnpsPLA(2)-IIa) noncovalently at submicromolar concentrations. Herein, the simple chiral precursor D-tyrosine was derivastised to give a series of potent new inhibitors of hnpsPLA(2)-IIa. A 2.2-Angstrom crystal structure shows an inhibitor bound in the active site of the enzyme, chelated to a Ca2+ ion through carboxylate and amide oxygen atoms, H bonded through an amide NH group to His48, with multiple hydrophobic contacts and a T-shaped aromatic-group-His6 interaction. Antiinflammatory activity is also demonstrated for two compounds administered orally to rats.

Benefits of ACE inhibitors with tissue-active levels in the cardiac-compromised patient

Angiotensin converting enzyme inhibitors (ACEI) have been proven beneficial to the cardiac-compromised patient, but whether there is an advantage associated with using a tissue-active or systemically-active ACEI is debatable. An investigation into the clinical benefits of tissue ACEI for veterinary patients was undertaken by comparing enalapril with ramipril. Results obtained concluded that although there is much evidence to prove that tissue ACEIs are superior over systemic ACEIs at the cellular level, this does not correlate in the clinical sense. Both enalapril and ramipril provided similar clinical benefits to the cardiac-compromised patient.

Histone-deacetylase inhibitors for the treatment of cancer

Histone deacetylase inhibitors (HDACi) are a promising new class of chemotherapeutic drug currently in early phase clinical trials. A large number of structurally diverse HDACi have been purified or synthesised that mostly inhibit the activity of all eleven class I and II HDACs. While these agents demonstrate many features required for anti-cancer activity such as low toxicity against normal cells and an ability to inhibit tumor cell growth and survival at nanomolar concentrations, their mechanisms of action are largely unknown. Initially, a model was proposed whereby HDACi-mediated transactivation of a specific gene or set of genes was responsible for the inhibition of cell cycle progression or induction of apoptosis. Given that HDACs can regulate the activity of a number of nonhistone proteins and that histone acetylation is important for events such as DNA replication and mitosis that do not directly involve gene transcription, it appears that the initial mechanistic model for HDACi may have been too simple. Herein, we provide an update on the transcription-dependent and - independent events that may be important for the anti-tumor activities of HDACi and discuss the use of these compounds in combination with other chemotherapeutic drugs.

Defining the chemotherapeutic targets of Histone Deacetylase inhibitors

The use of many conventional chemotherapeutic drugs is often severely restricted due to dose-limiting toxicities, as these drugs target the destruction of the proliferating fraction of cells, often with little specificity for tumor cells over proliferating normal body tissue. Many newer drugs attempt to overcome this shortcoming by targeting defective gene products or cellular mechanisms that are specific to the tumor, thereby minimizing the toxicity to normal tissue. Histone deacetylase inhibitors are an example of this type of tumor-directed drug, having significant toxicity for tumors but minimal effects on normal tissue. These drugs can affect the transcriptional program by modifying chromatin structure, but it is not yet clear whether specific transcriptional changes are directly responsible for their tumor-selective toxicity. Recent evidence suggests that transcriptional changes underlie their cytostatic activity, although this is not tumor-selective and affects all proliferating cells. Here we present evidence that supports an alternative mechanism for the tumor-selective cytotoxicity of histone deacetylase inhibitors. The target is still likely to be the chromatin histones, but rather than transcriptional changes due to modification of the transcriptionally active euchromatin, we propose that hyperacetylation and disruption of the transcriptionally inactive heterochromatin, particularly the centromeric heterochromatin, and the inability of tumor cells to cell cycle arrest in response to a specific checkpoint, underlies the tumor-selective cytotoxicity of these drugs.