Alejandra Moure

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.

A chemical inhibitor of Apaf-1 exerts mitochondrioprotective functions and interferes with the intra-S-phase DNA damage checkpoint

QM31 represents a new class of cytoprotective agents that inhibit the formation of the apoptosome, the caspase activation complex composed by Apaf-1, cytochrome c, dATP and caspase-9. Here, we analyzed the cellular effects of QM31, as compared to the prototypic caspase inhibitor Z-VAD-fmk. QM31 was as efficient as Z-VAD-fmk in suppressing caspase-3 activation, and conferred a similar cytoprotective effect. In contrast to Z-VAD-fmk, QM31 inhibited the release of cytochrome c from mitochondria, an unforeseen property that may contribute to its pronounced cytoprotective activity. Moreover, QM31 suppressed the Apaf-1-dependent intra-S-phase DNA damage checkpoint. These results suggest that QM31 can interfere with the two known functions of Apaf-1, namely apoptosome assembly/activation and intra-S-phase cell cycle arrest. Moreover, QM31 can inhibit mitochondrial outer membrane permeabilization, an effect that is independent from its action on Apaf-1.

Efficient Synthesis of Pseudopeptidic Molecular Cages

Pseudopeptidic cages have been efficiently prepared by combining a dynamic covalent procedure with the suitable preorganization of the building blocks by a conformational bias or an anion templation.

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-phase-based 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 molecular modeling 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.

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-κB 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-κB by TNF-α 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.

Pseudopeptidic Cages as Receptors for N-Protected Dipeptides

The molecular recognition of short peptides is a challenge in supramolecular chemistry, and the use of peptide-like cage receptors represents a promising approach. Here we report the synthesis and characterization of a diverse family of pseudopeptidic macrobicycles, as well as their binding abilities toward N-protected dipeptides using a combination of different techniques (NMR, ESI-MS, and fluorescence spectroscopy). The cage hosts were assayed for dipeptide binding using competition ESI-MS experiments as high-throughput screening to obtain general trends for the recognition phenomena. Selected hosts were additionally studied by NMR spectroscopy ((1)H NMR titration and diffusion-ordered spectroscopy experiments) in different solvents. The results unambiguously demonstrated the formation of the [cage·dipeptide] supramolecular complexes and rendered quantitative information about the strength of the interaction (K(ass)). The structural variables within the pseudopeptidic cage framework that produced a stronger and more selective recognition were thus identified. The cages showed a remarkable selectivity for N-protected dipeptides with an aromatic amino acid at the carboxylic terminus, which prompted us to propose a mode of binding based on polar and nonpolar noncovalent interactions. Accordingly, we faced the molecular recognition of a target dipeptide (Ac-EY-OH) mimicking a biologically relevant sequence by NMR and fluorescence spectroscopy in highly competitive media.

Triazine-Based Vanilloid 1 Receptor Open Channel Blockers: Design, Synthesis, Evaluation, and SAR Analysis

The thermosensory transient receptor potential vanilloid 1 channel (TRPV1) is a polymodal receptor activated by physical and chemical stimuli. TRPV1 activity is drastically potentiated by proinflammatory agents released upon tissue damage. Given the pivotal role of TRPV1 in human pain, there is pressing need for improved TRPV1 antagonists, the development of which will require identification of new pharmacophore scaffolds. Uncompetitive antagonists acting as open-channel blockers might serve as activity-dependent blockers that preferentially modulate the activity of overactive channels, thus displaying fewer side effects than their competitive counterparts. Herein we report the design, synthesis, biological evaluation, and SAR analysis of a family of triazine-based compounds acting as TRPV1 uncompetitive antagonists. We identified the triazine 8aA as a potent, pure antagonist that inhibits TRPV1 channel activity with nanomolar efficacy and strong voltage dependency. It represents a new class of activity-dependent TRPV1 antagonists and may serve as the basis for lead optimization in the development of new analgesics.

Salt-Induced Adaptation of a Dynamic Combinatorial Library of Pseudopeptidic Macrocycles: Unraveling the Electrostatic Effects in Mixed Aqueous Media

Dynamic combinatorial libraries are powerful systems for studying adaptive behaviors and relationships, as models of more complex molecular networks. With this aim, we set up a chemically diverse dynamic library of pseudopeptidic macrocycles containing amino-acid side chains with differently charged residues (negative, positive, and neutral). The responsive ability of this complex library upon the increase of the ionic strength has been thoroughly studied. The families of the macrocyclic members concentrating charges of the same sign showed a large increase in its proportion as the ionic strength increases, whereas those with residues of opposite charges showed the reverse behavior. This observation suggested an electrostatic shielding effect of the salt within the library of macrocycles. The top-down deconvolution of the library allowed us to obtain the fundamental thermodynamic information connecting the library members (exchange equilibrium constants), as well as to parameterize the adaptation to the external stimulus. We also visualized the physicochemical driving forces for the process by structural analysis using NMR spectroscopy and molecular modeling. This knowledge permitted the full understanding of the whole dynamic library and also the de novo design of dynamic chemical systems with tailored co-adaptive relationships, containing competing or cooperating species. This study highlights the utility of dynamic combinatorial libraries in the emerging field of systems chemistry.

Apaf-1 inhibitors protect from unwanted cell death in in vivo models of kidney ischemia and chemotherapy induced ototoxicity

Background Excessive apoptosis induces unwanted cell death and promotes pathological conditions. Drug discovery efforts aimed at decreasing apoptotic damage initially targeted the inhibition of effector caspases. Although such inhibitors were effective, safety problems led to slow pharmacological development. Therefore, apoptosis inhibition is still considered an unmet medical need. Methodology and Principal Findings The interaction between Apaf-1 and the inhibitors was confirmed by NMR. Target specificity was evaluated in cellular models by siRNa based approaches. Cell recovery was confirmed by MTT, clonogenicity and flow cytometry assays. The efficiency of the compounds as antiapoptotic agents was tested in cellular and in vivo models of protection upon cisplatin induced ototoxicity in a zebrafish model and from hypoxia and reperfusion kidney damage in a rat model of hot ischemia. Conclusions Apaf-1 inhibitors decreased Cytc release and apoptosome-mediated activation of procaspase-9 preventing cell and tissue damage in ex vivo experiments and in vivo animal models of apoptotic damage. Our results provide evidence that Apaf-1 pharmacological inhibition has therapeutic potential for the treatment of apoptosis-related diseases.

Synthesis of enantiomerically pure perhydro-1,4-diazepine-2,5-dione and 1,4-piperazine-2,5-dione derivatives exhibiting potent activity as apoptosis inhibitors

Apoptosis is the process of programmed cell death and plays a fundamental role in several human diseases. We have previously reported the synthesis of the perhydro-1,4-diazepine-2,5-dione and 1,4-piperazine-2,5-dione derivatives as racemic mixtures. Compounds 1 and 2 showed a potent in vitro and in cellular extracts antiapoptotic activity. In view that the chiral discrimination has been an issue in the development and use of pharmaceutical drugs, the present contribution reports the synthesis of enantiopure peptidomimetics 1 and 2. The biological evaluation of these enantiomers as apoptosis inhibitors is also reported.