Conjugation of a Novel Apaf-1 Inhibitor to Peptide-based Cell-Membrane Transporters. Effective Methods to Improve Inhibition of Mitochondria-Mediated Apoptosis

The definitive verison is available at http://www.sciencedirect.com/science//journal/01969781We have identified a family of peptoids that inhibits in vitro the activity of the apoptosome, a macromolecular complex that activates mitochondrial-dependent apoptosis pathways. The analysis of peptide-based cell compatible delivery systems of the most active peptoid is presented. The active peptoid was then fused to cell penetrating peptides (CPP) as penetratin (PEN-peptoid) and HIV-1 TAT (TAT-peptoid). PEN-peptoid showed greater cell viability and as a consequence better efficiency as an apoptosis inhibitor than the TAT-peptoid. The intracellular trafficking of both inhibitors was studied by flow cytometry and confocal fluorescence microscopy. Finally, the influence of the cargo (peptoid) molecules on the conformational behavior of the CPP in buffers and in membrane mimetic environments was analyzed using circular dichroism (CD) spectroscopy.This work was supported by grants from Spanish Ministry of Science and Education (MEC) (BIO2004-998 and CTQ2005-00995), Fundación Centro de Investigación Príncipe Felipe and a Marie Curie Reintegration grant (MERG-2004-06307). Mar Orzáez thanks Bancaja for a postdoctoral fellowship. Laura Mondragón is supported by a FPI fellowship from MEC. The confocal microscope was supported by MEC through FEDER.Peer reviewe

Compuestos para la inhibición de la apoptosis

La presente invención se refiere a compuestos que actúan como inhibidores de la apoptosis, así como a procedimientos para su preparación, a composiciones farmacéuticas que los contienen y a su uso en medicina.Peer reviewedLaboratorios SALVAT SA, Consejo Superior de Investigaciones Científicas (España)T3 Traducción de patente europe

Chemical Modulation of Peptoids: Synthesis and Conformational Studies on Partially Constrained Derivatives

The high conformational flexibility of peptoids can generate problems in biomolecular selectivity as a result of undesired off-target interactions. This drawback can be counterbalanced by restricting the original flexibility to a certain extent, thus leading to new peptidomimetics. By starting from the structure of an active peptoid as an apoptosis inhibitor, we designed two families of peptidomimetics that bear either 7-substituted perhydro- 1,4-diazepine-2,5-dione 2 or 3-substituted 1,4-piperazine-2,5-dione 3 moieties. We report an efficient, solid-phasebased synthesis for both peptidomimetic families 2 and 3 from a common intermediate. An NMR spectroscopic study of 2a,b and 3a,b showed two species in solution in different solvents that interconvert slowly on the NMR timescale. The cis/trans isomerization around the exocyclic tertiary amide bond is responsible for this conformational behavior. The cis isomers are more favored in nonpolar environments, and this preference is higher for the six-membered-ring derivative 3a,b. We propose that the hydrogen-bonding pattern could play an important role in the cis/trans equilibrium process. These hydrogen bonds were characterized in solution, in the solid state (i.e., by using X-ray studies), and by molecularmodeling of simplified systems. A comparative study of a model peptoid 10 containing the isolated tertiary amide bond under study outlined the importance of the heterocyclic moiety for the prevalence of the cis configuration in 2a and 3a. The kinetics of the cis/trans interconversion in 2a, 3a, and 10 was also studied by variable-temperature NMR spectroscopic analysis. The full line-shape analysis of the NMR spectra of 10 revealed negligible entropic contribution to the energetic barrier in this conformational process. A theoretical analysis of 10 supported the results observed by NMR spectroscopic analysis. Overall, these results are relevant for the study of the peptidomimetic/biological- target interactions.Support from MICINN (Grants CTQ2005-00995, SAF2008-00048, BIO2007 60066) is acknowledged. The authors thank F. J. Morales and M. Camargo for technical assistance. A JAE-CSIC fellowship to A.M. is also acknowledged.Peer reviewe

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

10 pages, 5 figures.-- PMID: 16341125 [PubMed].-- Available online Dec 9, 2005.Supporting information available at: http://www.nature.com/cdd/journal/v13/n9/suppinfo/4401828s1.html?url=/cdd/journal/v13/n9/abs/4401828a.htmlApoptosis 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.This work was supported by grants from Spanish Ministry of Science and Techonology (SAF2001-2811, SAF2001-2286 and BIO2004-998), Fundación Areces and Fundación Valenciana de Investigaciones Biomédicas.Peer reviewe

Polymer conjugate compounds for inhibition of apoptosis

Fecha de presentación internacional 21.05.2007.-- Titulares: Laboratorios Salvat, S.A., Consejo Superior de Investigaciones Científicas.The present invention relates to polymer cdonjugates compounds, as well as processes for their preparation, to pharmatheutical compositions containing them and their use in medicine.Peer reviewe

Compuestos con actividad inhibidora de las interaciones UBC13-UEV, composiciones farmacéuticas y aplicaciones terapéuticas

Fecha de solicitud: 04-02-2009.- Titular: Consejo Superior de Investigaciones Científicas (CSIC)The invention relates to a compound R-(CR 1 R 2 )q-CO-N(R 3 )-C(R 4R 5 )-CO-NH 2 (I), wherein: R is a heterocyclyl radical; R 1 and R 2 independently represent H or alkyl; R 3 is H, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, aryl, arylalkyl, heterocyclyl or heterocyclylalkyl; R 4 and R 5 independently represent H or alkyl; q is a number between 0 and 1; and the salts, solvates, pro-drugs or stereoisomers thereof, which can inhibit UBC13-UEV interactions and be used in the production of pharmaceutical compositions intended for antitumour therapy or the treatment and/or prophylaxis of diseases associated with metabolic routes involving the UBC13 enzyme, metabolic routes involving transcriptional factor NF-kappaB or routes involving PCNA or RAD6.La invención relaciona a un r del compuesto (CR 1 R 2) q-CO-n (R 3) - C (R 4 R 5) - CO-NH 2 (i), en donde: R es un heterociclil radical R 1 y R 2 representan independientemente H o el alquílico R 3 es H, alquílico, cicloalquil, cicloalquilalquilo, alquenil, arílico, arilalquilo, heterociclil o heterocyclylalkyl R 4 y R 5 representan independientemente H o el alquílico q es un número entre 0 y 1 y las sales, los solvates, los pro-drugs o los stereoisomers de eso, que el bote inhibe interacciones de UBC13-UEV y sea utilizado en la producción de composiciones farmacéuticas destinadas para la terapia antitumour o el tratamiento y/o la profilaxis de las enfermedades asociadas con las vías metabólicas que implican la enzima UBC13, las vías metabólicas que implican el factor transcripcional N-F-kappaB o las vías que implican PCNA o RAD6.Peer reviewe

Protein-protein interaction antagonists as novel inhibitors of non-canonical polyubiquitylation

[Background]: Several pathways that control cell survival under stress, namely RNF8-dependent DNA damage recognition and repair, PCNA-dependent DNA damage tolerance and activation of NF-kB by extrinsic signals, are regulated by the tagging of key proteins with lysine 63-based polyubiquitylated chains, catalyzed by the conserved ubiquitin conjugating heterodimeric enzyme Ubc13-Uev. [Methodology/Principal Findings]: By applying a selection based on in vivo protein-protein interaction assays of compounds from a combinatorial chemical library followed by virtual screening, we have developed small molecules that efficiently antagonize the Ubc13-Uev1 protein-protein interaction, inhibiting the enzymatic activity of the heterodimer. In mammalian cells, they inhibit lysine 63-type polyubiquitylation of PCNA, inhibit activation of NF-kB by TNF-a and sensitize tumor cells to chemotherapeutic agents. One of these compounds significantly inhibited invasiveness, clonogenicity and tumor growth of prostate cancer cells. [Conclusions/Significance]: This is the first development of pharmacological inhibitors of non-canonical polyubiquitylation that show that these compounds produce selective biological effects with potential therapeutic applications.This work was funded by grants from the Consejo Superior de Investigaciones Cientificas (PIF200580 to T.M.T., A.R.O. and A. Messeguer), the Ministerio de Educacion y Ciencia (SAF2005-05109-CO2-01 to T.M.T.; CTQ2005-00995 and GEN2003-20642-C09-09 to A. Messeguer; BIO2005-0576, and GEN2003-206420-C09-08 to A.R.O.), the Comunidad de Madrid (GR/SAL/0306/2004 and 200520M157 to A.R.O.), and an institutional grant from the Fundacion Ramon Areces. J.S. and M.G.R. are receipients of fellowships from the Ministerio de Educacion y Ciencia, and A. Moure of a I3P fellowship from the Consejo Superior de Investigaciones Cientificas.Peer reviewe