Mar Orzáez

Small molecule inhibitors of Apaf-1-related caspase- 3/-9 activation that control mitochondrial-dependent apoptosis

Apoptosis is a biological process relevant to human disease states that is strongly regulated through protein–protein complex formation. These complexes represent interesting points of chemical intervention for the development of molecules that could modulate cellular apoptosis. The apoptosome is a holoenzyme multiprotein complex formed by cytochrome c-activated Apaf-1 (apoptotic protease-activating factor), dATP and procaspase-9 that link mitochondria disfunction with activation of the effector caspases and in turn is of interest for the development of apoptotic modulators. In the present study we describe the identification of compounds that inhibit the apoptosome-mediated activation of procaspase-9 from the screening of a diversity-oriented chemical library. The active compounds rescued from the library were chemically optimised to obtain molecules that bind to both recombinant and human endogenous Apaf-1 in a cytochrome c-noncompetitive mechanism that inhibits the recruitment of procaspase-9 by the apoptosome. These newly identified Apaf-1 ligands decrease the apoptotic phenotype in mitochondrial-mediated models of cellular apoptosis.

Modulation of Cellular Apoptosis with Apoptotic Protease-Activating Factor 1 (Apaf-1) Inhibitors

The programmed cell death or apoptosis plays both physiological and pathological roles in biology. Anomalous activation of apoptosis has been associated with malignancies. The intrinsic mitochondrial pathway of apoptosis activation occurs through a multiprotein complex named the apoptosome. We have discovered molecules that bind to a central protein component of the apoptosome, Apaf-1, and inhibits its activity. These new first-in-class apoptosome inhibitors have been further improved by modifications directed to enhance their cellular penetration to yield compounds that decrease cell death, both in cellular models of apoptosis and in neonatal rat cardiomyocytes under hypoxic conditions.

Temperature-controlled release by changes in the secondary structure of peptides anchored onto mesoporous silica supports

Changes in the conformation of a peptide anchored onto the external surface of mesoporous silica nanoparticles have been used to design novel temperature-controlled delivery systems.

Enzyme‐Responsive Silica Mesoporous Supports Capped with Azopyridinium Salts for Controlled Delivery Applications

The preparation of a new capped silica mesoporous material, Rh-Azo-S, for on-command delivery applications in the presence of target enzymes is described. The material consists of nanometric mesoporous MCM-41-like supports loaded with Rhodamine B and capped with an azopyridine derivative. The material was designed to show zero delivery and to display a cargo release in the presence of reductases and esterases, which are usually present in the colon, mainly due to intestinal microflora. The opening and cargo release of Rh-Azo-S in vitro studies were assessed and seen to occur in the presence of these enzymes, whereas no delivery was noted in the presence of pepsine. Moreover, Rh-Azo-S nanoparticles were used to study controlled Rhodamineu2005B dye delivery in intracellular media. HeLa cells were employed for testing the non-toxicity of nanoparticles. Moreover, delivery of the dye in these cells, through internalization and enzyme-mediated gate opening, was confirmed by confocal microscopy. Furthermore, the nanoparticles capped with the Azo group and loaded with a cytotoxic camptothecin (CPT) were also prepared (solid CPT-Azo-S) and used as delivery nanodevices in HeLa cells. When this solid was employed, the cell viability decreased significantly due to internalization of the nanoparticles and delivery of the cytotoxic agent.

Azobenzene Polyesters Used as Gate‐Like Scaffolds in Nanoscopic Hybrid Systems

The synthesis and characterisation of new capped silica mesoporous nanoparticles for on-command delivery applications is reported. Functional capped hybrid systems consist of MCM-41 nanoparticles functionalised on the external surface with polyesters bearing azobenzene derivatives and rhodamineu2005B inside the mesopores. Two solid materials, Rh-PAzo8-S and Rh-PAzo6-S, containing two closely related polymers, PAzo8 and PAzo6, in the pore outlets have been prepared. Materials Rh-PAzo8-S and Rh-PAzo6-S showed an almost zero release in water due to steric hindrance imposed by the presence of anchored bulky polyesters, whereas a large delivery of the cargo was observed in the presence of an esterase enzyme due to the progressive hydrolysis of polyester chains. Moreover, nanoparticles Rh-PAzo8-S and Rh-PAzo6-S were used to study the controlled release of the dye in intracellular media. Nanoparticles were not toxic for HeLa cells and endocytosis-mediated cell internalisation was confirmed by confocal microscopy. Furthermore, the possible use of capped materials as a drug-delivery system was demonstrated by the preparation of a new mesoporous silica nanoparticle functionalised with PAzo6 and loaded with the cytotoxic drug camptothecin (CPT-PAzo6-S). Following cell internalisation and lysosome resident enzyme-dependent gate opening, CPT-PAzo6-S induced CPT-dependent cell death in HeLa cells.

Intrinsic caspase-8 activation mediates sensitization of erlotinib-resistant tumor cells to erlotinib/cell-cycle inhibitors combination treatment

Inhibitors of the tyrosine kinase activity of epidermal growth factor receptor, as erlotinib, have an established role in treating several cancer types. However, resistance to erlotinib, particularly in breast cancer cell lines, and erlotinib treatment-associated disorders have also been described. Also, methods and combination therapies that could reverse resistance and ameliorate non-desirable effects represent a clinical challenge. Here, we show that the ATP non-competitive CDK2/cyclin A inhibitor NBI1 sensitizes erlotinib-resistant tumor cells to the combination treatment (co-treatment) for apoptosis-mediated cell death. Furthermore, in erlotinib-sensitive cells, the effective dose of erlotinib was lower in the presence of NBI1. The analysis in the breast cancer MDA-MB-468 erlotinib-resistant and in lung cancer A549 cell lines of the molecular mechanism underlying the apoptosis induced by co-treatment highlighted that the accumulation of DNA defects and depletion of cIAP and XIAP activates the ripoptosome that ultimately activates caspases-8 and -10 and apoptosis. This finding could have significant implications for future treatment strategies in clinical settings.

Discovery of Inhibitors of Protein-Protein Interactions from Combinatorial Libraries

Protein-protein interactions play a central role within numerous processes in the cell. The relevance of the processes in which this type of interactions are implicated make them responsible for many pathological situations. In the last decade protein-protein interfaces have shown their potential as new drug targets, and combinatorial chemistry has been defined as a useful tool in this line. This review gives a global vision of the actual situation of combinatorial chemistry, highlighting its applicability to high-throughput drug discovery and giving some crucial examples of its contribution to find modulators of protein-protein interactions.

L-Aminoacid Oxidase from Bothrops leucurus Venom Induces Nephrotoxicity via Apoptosis and Necrosis

Acute renal failure is a common complication caused by Bothrops viper envenomation. In this study, the nefrotoxicity of a main component of B. leucurus venom called L-aminoacid oxidase (LAAO-Bl) was evaluated by using tubular epithelial cell lines MDCK and HK-2 and perfused kidney from rats. LAAO-Bl exhibited cytotoxicity, inducing apoptosis and necrosis in MDCK and HK-2 cell lines in a concentration-dependent manner. MDCK apoptosis induction was accompanied by Ca2+ release from the endoplasmic reticulum, reactive oxygen species (ROS) generation and mitochondrial dysfunction with enhanced expression of Bax protein levels. LAAO-Bl induced caspase-3 and caspase-7 activation in both cell lines. LAAO-Bl (10 μg/mL) exerts significant effects on the isolated kidney perfusion increasing perfusion pressure and urinary flow and decreasing the glomerular filtration rate and sodium, potassium and chloride tubular transport. Taken together our results suggest that LAAO-Bl is responsible for the nephrotoxicity observed in the envenomation by snakebites. Moreover, the cytotoxic of LAAO-Bl to renal epithelial cells might be responsible, at least in part, for the nephrotoxicity observed in isolated kidney.

Cyclin-Dependent Kinase (CDK) Inhibitors

Cyclin-dependent kinases (Cdks) belong to a family of key regulators of cell division cycle and transcription. The activity of some of them is deregulated in tumor cells and to fi nd specifi c inhibitors is an important goal to be achieved. We report here the current methods to determine their in vitro activity in order to facilitate the identifi cation of specifi c inhibitors. Mainly, the activity can be determined by using immunoprecipitates from cell samples with antibodies against specifi c Cdks as a source of the enzymes.

Helix-Helix Packing Between Transmembrane Fragments

The rules that govern the folding of membrane proteins are still not completely understood when compared with the well-detailed set of principles described for the folding of soluble proteins. Although the molecular determinants of the folding mechanism should be basically the same for both types of proteins, the main difference, which in turn could also represent the main difficulty, is the media that surrounds the protein. In this sense, it would be useful to develop new molecular techniques that allow for the study of the folding mechanism of membrane proteins in their natural media, the membrane. In the present work we have collected a series of studies devoted to understanding the principles that govern the molecular mechanism of folding and packing of membrane proteins, an important group of the proteome. In particular, we have focused our attention on Glycophorin A (GpA), a single span membrane protein, which has been extensively used as a model system to study helix-helix transmembrane (TM) packing. Here, we report on the importance of the molecular distance between the critical oligomerisation motif and polar residues in TM fragments. We have also given an account of the influence of the length of the TM fragment as well as the effect of proline residues on the packing of TM alpha helices.