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Compuestos para el tratamiento de enfermedades causadas por Helicobacter. La presente invención se refiere a nuevos compuestos que son benzo [c] [1,2,5] oxadiazol. derivados, y su uso en el tratamiento de enfermedades infecciosas causadas por Helicobacter pylori. Además, la presente invención se refiere a una composición farmacéutica y a una preparación combinada que comprende dichos compuestos.

Hereditary transthyretin amyloidosis (ATTR) is a disease characterized by the extracellular deposition of transthyretin (TTR) amyloid fibrils. Highly destabilizing TTR mutations cause leptomeningeal amyloidosis, a rare, but fatal, disorder in which TTR aggregates in the brain. The disease remains intractable, since liver transplantation, the reference therapy for systemic ATTR, does not stop mutant TTR production in the brain. In addition, despite current pharmacological strategies have shown to be effective against in vivo TTR aggregation by stabilizing the tetramer native structure and precluding its dissociation, they display low brain permeability. Recently, we have repurposed tolcapone as a molecule to treat systemic ATTR. Crystal structures and biophysical analysis converge to demonstrate that tolcapone binds with high affinity and specificity to three unstable leptomeningeal TTR variants, stabilizing them and, consequently, inhibiting their aggregation. Because tolcapone is an FDA-approved drug that crosses the blood-brain barrier, our results suggest that it can translate into a first disease-modifying therapy for leptomeningeal amyloidosis. DATABASES: PDB codes for A25T-TTR, V30G-TTR, and Y114C-TTR bound to tolcapone are 6TXV, 6TXW, and 6XTK, respectively.

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-binding protein involved in neuronal development and maturation. Loss-of-function mutations in MeCP2 result in Rett syndrome (RTT), a neurodevelopmental disorder that is the main cause of mental retardation in females. MeCP2 is an intrinsically disordered protein (IDP) constituted by six domains. Two domains are the main responsible elements for DNA binding (methyl-CpG binding domain, MBD) and recruitment of gene transcription/silencing machinery (transcription repressor domain, TRD). These two domains concentrate most of the RTT-associated mutations. R106W and R133C are associated with severe and mild RTT phenotype, respectively. We have performed a comprehensive characterization of the structural and functional impact of these substitutions at molecular level. Because we have previously shown that the MBD-flanking disordered domains (N-terminal domain, NTD, and intervening domain, ID) exert a considerable influence on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41635), here we report the biophysical study of the influence of the protein scaffold on the structural and functional effect induced by these two RTT-associated mutations. These results represent an example of how a given mutation may show different effects (sometimes opposing effects) depending on the molecular context.

Establishing the interactome of the cancer-associated stress protein Nuclear Protein 1 (NUPR1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair, and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1, thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further report that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system, indicating that NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation-dependent functions of key proteins involved in the DDR.

Background: Current efforts in the identification of new biomarkers are directed towards an accurate differentiation between benign and premalignant cysts. Thermal Liquid Biopsy (TLB) has been previously applied to inflammatory and tumor diseases and could offer an interesting point of view in this type of pathology. Methods: In this work, twenty patients (12 males and 8 females, average ages 62) diagnosed with a pancreatic cyst benign (10) and premalignant (10) cyst lesions were recruited, and biological samples were obtained during the endoscopic ultrasonography procedure. Results: Proteomic content of cyst liquid samples was studied and several common proteins in the different groups were identified. TLB cyst liquid profiles reflected protein content. Also, TLB serum score was able to discriminate between healthy and cysts patients (71% sensitivity and 98% specificity) and between benign and premalignant cysts (75% sensitivity and 67% specificity). Conclusions: TLB analysis of plasmatic serum sample, a quick, simple and non-invasive technique that can be easily implemented, reports valuable information on the observed pancreatic lesion. These preliminary results set the basis for a larger study to refine TLB serum score and move closer to the clinical application of TLB providing useful information to the gastroenterologist during patient diagnosis.

Nucleoplasmin (NP) is a pentameric histone chaperone that regulates the condensation state of chromatin in different cellular processes. We focus here on the interaction of NP with the histone octamer, showing that NP could bind sequentially the histone components to assemble an octamer-like particle, and crosslinked octamers with high affinity. The three-dimensional reconstruction of the NP/octamer complex generated by single-particle cryoelectron microscopy, revealed that several intrinsically disordered tail domains of two NP pentamers, facing each other through their distal face, encage the histone octamer in a nucleosome-like conformation and prevent its dissociation. Formation of this complex depended on post-translational modification and exposure of the acidic tract at the tail domain of NP. Finally, NP was capable of transferring the histone octamers to DNA in vitro, assembling nucleosomes. This activity may have biological relevance for processes in which the histone octamer must be rapidly removed from or deposited onto the DNA.

Risk population screening programs are instrumental for advancing cancer management and reducing economic costs of therapeutic interventions and the burden of the disease, as well as increasing the survival rate and improving the quality of life for cancer patients. Lung cancer, with high incidence and mortality rates, is not excluded from this situation. The success of screening programs relies on many factors, with some of them being the appropriate definition of the risk population and the implementation of detection techniques with an optimal discrimination power and strong patient adherence. Liquid biopsy based on serum or plasma detection of circulating tumor cells or DNA/RNA is increasingly employed nowadays, but certain limitations constrain its wide application. In this work, we present a new implementation of thermal liquid biopsy (TLB) for lung cancer patients. TLB provides a prediction score based on the ability to detect plasma/serum proteome alterations through calorimetric thermograms that strongly correlates with the presence of lung cancer disease (91% accuracy rate, 90% sensitivity, 92% specificity, diagnostic odds ratio 104). TLB is a quick, minimally-invasive, low-risk technique that can be applied in clinical practice for evidencing lung cancer, and it can be used in screening and monitoring actions.

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their prevalence in various diseases including cancer. Drug development targeting IDPs is challenging because they have dynamical structure features and conventional drug design is not applicable. NUPR1 is an IDP playing an important role in pancreatic cancer. We previously reported that Trifluoperazine (TFP), an antipsychotic agent, was capable of binding to NUPR1 and inhibiting tumors growth. Unfortunately, TFP showed strong central nervous system side-effects. In this work, we undertook a multidisciplinary approach to optimize TFP, based on the synergy of computer modeling, chemical synthesis, and a variety of biophysical, biochemical and biological evaluations. A family of TFP-derived compounds was produced and the most active one, named ZZW-115, showed a dose-dependent tumor regression with no neurological effects and induced cell death mainly by necroptosis. This study opens a new perspective for drug development against IDPs, demonstrating the possibility of successful ligand-based drug design for such challenging targets.

Cancer cells activate stress-response mechanisms to adapt themselves to a variety of stressful conditions. Among these protective mechanisms, those controlled by the stress-induced nuclear protein 1 (NUPR1 ) belong to the most conserved ones. NUPR1 is an 82-residue-long, monomeric, basic and intrinsically disordered protein (IDP), which was found to be invariably overexpressed in some, if not all, cancer tissues. Remarkably, we and others have previously showed that genetic inactivation of the Nupr1 gene antagonizes the growth of pancreatic cancer as well as several other tumors. With the use of a multidisciplinary strategy by combining biophysical, biochemical, bioinformatic, and biological approaches, a trifluoperazine-derived compound, named ZZW-115, has been identified as an inhibitor of the NUPR1 functions. The anticancer activity of the ZZW-115 was first validated on a large panel of cancer cells. Furthermore, ZZW-115 produced a dose-dependent tumor regression of the tumor size in xenografted mice. Mechanistically, we have demonstrated that NUPR1 binds to several importins. Because ZZW-115 binds NUPR1 through the region around the amino acid Thr68, which is located into the nuclear location signal (NLS) region of the protein, we demonstrated that treatment with ZZW-115 inhibits completely the translocation of NUPR1 from the cytoplasm to the nucleus by competing with importins.

Brucella ovis encodes a bacterial subclass 1 ferredoxin-NADP(H) reductase (BoFPR) that, by similarity with other FPRs, is expected either to deliver electrons from NADPH to the redox-based metabolism and/or to oxidize NADPH to regulate the soxRS regulon that protects bacteria against oxidative damage. Such potential roles for the pathogen survival under infection conditions make of interest to understand and to act on the BoFPR mechanism. Here, we investigate the NADP/H interaction and NADPH oxidation by hydride transfer (HT) to BoFPR. Crystal structures of BoFPR in free and in complex with NADP hardly differ. The latter shows binding of the NADP adenosine moiety, while its redox-reactive nicotinamide protrudes towards the solvent. Nonetheless, pre-steady-state kinetics show formation of a charge-transfer complex (CTC-1) prior to the hydride transfer, as well as conversion of CTC-1 into a second charge-transfer complex (CTC-2) concomitantly with the HT event. Thus, during catalysis nicotinamide and flavin reacting rings stack. Kinetic data also identify the HT itself as the rate limiting step in the reduction of BoFPR by NADPH, as well as product release limiting the overall reaction. Using all-atom molecular dynamics simulations with a thermal effect approach we are able to visualise a potential transient catalytically competent interaction of the reacting rings. Simulations indicate that the architecture of the FAD folded conformation in BoFPR might be key in catalysis, pointing to its adenine as an element to orient the reactive atoms in conformations competent for HT.

Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation.

Biological systems are the result of the interactions established among their many distinct molecules and molecular assemblies. The high concentration of small molecules dissolved in the aqueous media alter the water properties with important consequences in the interactions established. In this work, the effects of high concentrations of the disaccharide trehalose on the solubility of a homologous series of fluorescent amphiphiles (NBD-Cn, n=4–16) and on their interaction with a lipid bilayer and a serum protein are quantitatively characterized. Both kinetic and equilibrium aspects are reported for a better understanding of the effects observed. The aqueous solubility of the most hydrophobic amphiphiles (n ≥ 8) is strongly increased by 1 M trehalose, while no signifcant effect is observed for the most polar amphiphile (n = 4). This results from a decrease in the magnitude of the hydrophobic effect at molecular crowding conditions. A small decrease is observed on the equilibrium association with serum albumin. This is most significant for amphiphiles with longer alkyl chains, in agreement with their increased solubility in the aqueous media containing trehalose. The effects on the association of the amphiphiles with lipid bilayers are influenced by both equilibrium and kinetic aspects. On the one hand, the decreased magnitude of the hydrophobic effect leads to a decrease in the affinity of the amphiphiles towards the membrane. However, this tendency may be overbalanced by the effects on the kinetics of the interaction (insertion/desorption) due to the increase in the viscosity of the aqueous media. It is shown that the distribution of amphiphilic drugs in the crowded biological media is significantly different from that predicted from studies in dilute solutions and that the effects are dependent on the solute's hydrophobicity.

Intrinsically disordered proteins (IDPs) do not have a well-defined structure under physiological conditions, but they have key roles in cell signaling and regulation, and they are frequently related to the development of diseases, such as cancer and other malignancies. This has converted IDPs in attractive therapeutic targets; however, targeting IDPs is challenging because of their dynamic nature. In the last years, different experimental and computational approaches, as well as the combination of both, have been explored to identify molecules to target either the hot-spots or the allosteric sites of IDPs. In this review, we summarize recent developments in successful targeting of IDPs, all of which are involved in different cancer types. The strategies used to develop and design (or in one particular example, to repurpose) small molecules targeting IDPs are, in a global sense, similar to those used in well-folded proteins: (1) screening of chemically diverse or target-oriented compound libraries; or (2) study of the interfaces involved in recognition of their natural partners, and design of molecular candidates capable of binding to such binding interface. We describe the outcomes of using these approaches in targeting IDPs involved in cancer, in the view to providing insight, to target IDPs in general. In a broad sense, the designed small molecules seem to target the most hydrophobic regions of the IDPs, hampering macromolecule (DNA or protein)–IDP interactions; furthermore, in most of the molecule–IDP complexes described so far, the protein remains disordered.

Biological processes rely on interactions between many binding partners. Binding results in the modulation of the conformational landscape of the interacting molecules, a phenomenon rooted in folding and binding cooperativity underlying the allosteric functional regulation of biomacromolecules. The conformational equilibrium of a protein and the binding equilibria of different interacting and cooperative ligands are coupled giving rise to a complex scenario in which protein function can be finely tuned and modulated. Binding cooperativity and allostery add additional levels of complexity in protein function regulation. Here we will review some important concepts associated with binding, cooperativity and allostery in protein interactions, illustrated with several representative protein-dependent biological systems related to drug discovery and physiological mechanisms characterization and studied by isothermal titration calorimetry.