Ayuda
Ir al contenido

Dialnet


Estudios de interacciones macromoleculares en el diseño de antivirales y antibióticos

  • Autores: Rosa María Doménech Mata
  • Directores de la Tesis: Jose Luis Neira Falairo (dir. tes.)
  • Lectura: En la Universidad Miguel Hernández ( España ) en 2012
  • Idioma: español
  • Tribunal Calificador de la Tesis: Javier Sancho Sanz (presid.), Luis Pérez García-Estañ (secret.), Ernesto Cota (voc.), Ana María Cámara Artigas (voc.), Ana Isabel Azuaga Fortes (voc.)
  • Materias:
  • Texto completo no disponible (Saber más ...)
  • Resumen
    • Global ecology alterations, technology and human population growth go together with the evolutionary change in other species, especially in pathogenic organisms. Bacterial diseases have evolved strong and devastating resistance to many antibiotics and, in some cases, bacteria have been able to develop simultaneous resistance to more than one antibiotic. Treatments that required small antibiotic doses, now require huge concentrations, or demand powerful new drugs (Garrett, 1994). Moreover, retroviruses with RNA genomes evolve even more quickly than bacteria (Crandall, 1999). Furthermore, mutations in the HIV reverse transcriptase gene quickly arise, confering drug resistance to the HIV.

      In order to find effective drugs to such resistances, we proposed the design of compounds capable of inhibiting the protein-protein recognition interfaces in: i) the dimerization interface of the capsid protein of HIV-1 (CA) for the research of new anti-VIH drugs; and, ii) the phosphotransferase system (PTS), specifically the enzyme I and HPr, the non-sugar specific proteins of the PTS in Streptomyces coelicolor, for the development of new antibiotic drugs. For the sake of the reader, we develop two sections: Search for new anti-VIH drugs Virus capsid assembly constitutes an attractive target for the development of antiviral therapies. Assembly of the mature HIV-1 capsid involves the oligomerization of the capsid protein, CA. During retroviral maturation, CA undergoes structural changes and forms exclusive intermolecular interfaces in the mature capsid shell, different from those in the immature precursor. CA is a 26 kDa dimeric protein and it is formed by two independently folded domains, separated by a flexible linker (Gamble et al., 1996, 1997): i) the N-terminal domain, CAN (residues 1¿146 ), is a monomer and it is composed of five ¿-helices (corresponding to ¿-helices 1¿5) with two additional short ¿-helices (¿-helices 6 and 7) following an extended proline-rich loop; and, ii) the C-terminal one, CAC (residues 147¿231) which dimerizes in solution with similar affinity as the intact CA (~15 ¿M [Gamble et al., 1997]), participates both in the formation of CA hexamers and in the joining of hexamers through homodimerization. Each CAC monomer is composed of a short 310-helix followed by a strand and four ¿-helices connected by short loops or turn-like structures: ¿-helix 8 (residues 160¿172), the dimerization ¿-helix 9 (residues 178¿191), ¿-helix 10 (residues 195¿202) and ¿-helix 11 (residues 209¿214). The polypeptide region formed by residues Asp152 to Leu172 is called the major homology region, MHR; it is highly conserved among all the retroviruses and it is involved in both immature and mature virus assembly. However, its exact function during both assembly stages remains unknown.

      The present work has focused on two goals: i) the inhibition of CA assembly based on the design of interfacial inhibitors of the dimerization interface: these compounds are small organic compounds and peptide-mimetics of the dimerization interface (¿-helix 9); and, ii) determining the role of MHR during CA assembly, by designing a peptide which comprises the whole region.

      In the first part of this work we have rationally designed and/or modified different interfacial peptides that represent the ¿-helix 9 that could act as competitive assembly inhibitors. These are derived from CAC1, a previously designed peptide which contains a major part of the dimerization interface: i) Two peptides were designed to increase the solubility of CAC1 and the CA-binding affinity (CAC1C and CAC1M). We mapped the binding site of CAC1, CAC1C and CAC1M, and shown that it substantially overlaps with the CAC dimerization interface. We found that CAC1 and its derivative CAC1M were able to efficiently inhibit the assembly in vitro of CA. So, both peptides could serve as lead compounds for development of anti-HIV agents.

      ii) Four peptides were designed with predicted higher helical propensities than the wild-type sequence, while keeping important residues for dimerization (P1-P4). Our results revealed that these peptides showed a higher helicity than that of the wild-type peptide, although not as high as theoretically predicted. They are able to self-associate with similar affinities to that of CAC. However, binding to CAC mainly occurs at the last ¿-helical region of the protein (¿-helix 11); accordingly, most of those peptides are unable to inhibit CA polymerization in vitro. P1 was the only peptide which presented inhibitory activity, but not at a high extent. It is worth saying that this designed peptide has the maximal helical content achieved by the wild-type sequence of the interface.

      In the second part of this work we have explored the binding process of the first-generation gallic acid-triethylene glycol (GATG) dendrimers to CAC. Our results revealed that, the binding region is mainly formed by residues involved in the homodimerization interface of CAC. The affinity for CAC of some of the dendrimers is similar to that of synthetic peptides capable of binding to the dimerization region (Garzón et al., 2004) and its derivatives, and it is also similar to the homodimerization affinity of both CAC and CA. Moreover, only the molecule with a benzoate moiety at the dendrimer branching, [G1]-CO2Na, was able to hamper the CA assembly in vitro. These results suggest that dendrimers could be potential compounds for the development of anti-VIH drugs, targeting capsid assembly.

      Finally, in the third part of this work we have used a peptide comprising the MHR region, namely MHRpep (residues Asp152 to Ala174). Our results revealed that isolated MHRpep is mainly unfolded in aqueous solution, with residual structure at its C terminus. MHRpep binds to monomeric CAC (~30 ¿M), as shown by fluorescence and ITC experiments; the CAC binding region comprises residues belonging to the last two ¿-helices of CA (¿-helix 10 and ¿-helix 11). In the immature virus capsid, the MHR region and the ¿-helix 11 of two CAC dimers also interact (Briggs et al., 2009). These results can be considered a proof-of-concept that the conformational preferences and the binding features of isolated peptides derived from virus proteins could be used to mimic early stages of virus assembly.

      Design of new antibiotics The phosphotransferase system (PTS) is involved in the use of carbon sources in bacteria. The PTS catalyses the transfer of an activated phosphoryl group from phosphoenolpyruvate (PEP) to the imported carbohydrate. This catalysis occurs through a cascade of five proteins from PEP: two general proteins, namely enzyme I (EI) and the His-phosphocarrier protein (HPr), and various sugar-specific permeases. EI autophosphorylates in the presence of PEP and Mg2+. It is an homodimer with a molecular weight of 60 kDa and it is formed of two domains: the N-terminal domain, EIN, which comprises the HPr-binding site and the phosphorylation acive site-His; and the C-terminal domain, EIC, which is responsible for the dimerization and where the PEP-binding domain is located. HPr is a 9 kDa monomeric protein. In low-G+C gram-positive bacteria, HPr becomes phosphorylated not only by phosphoenolpyruvate (PEP) at the active site-His, but also by ATP at the regulatory Ser.

      The PTS is ubiquitous in bacteria, but it does not occur in plants and animals; it modulates catabolite repression, intermediate metabolism, gene expression and chemotaxis. Its uniqueness and pleiotropic function make the PTS, and specially EIN and HPr, an attractive target for new antibacterial drugs.

      In this work, we have firstly studied the stability and binding of EIN from S. coelicolor (EINsc) to HPr from S. coelicolor (HPrsc) in their phosphorylated states to determine the effect of the phosphorylation in the stability and affinity among these non-sugar specific proteins. For that purpose, we have characterized the stability and binding affinities of: i) the active site-His phosphorylated species of EINsc (at His186) and HPrsc (at His15); and, ii) the species involving the phosphorylation at the regulatory Ser of HPrsc (Ser47). Our results show that the phosphorylated active-site species of both proteins are less stable than the unphosphorylated counterparts. Conversely, the HPrS47D, which mimics phosphorylation at Ser47, is more stable than wild-type HPrsc due to helical N-capping effects, as suggested by the modelled structure of the protein (Poveda et al., 2007). Binding among the phosphorylated and unphosphorylated species is always entropically driven, but the affinity and the enthalpy vary widely. From these results, we suggest that the dimerization of the EICsc is important in determining the affinity of the intact EIsc for HPrsc.

      Furthermore, in our search for an inhibitor of the PTS system, hampering the binding of EINsc to HPrsc, we have used peptides derived from EINsc against HPrsc and assayed the binding affinities; they bound to the region close to the active site-histidine. Furthermore, we assayed the affinity of EINsc by: (i) peptides derived from HPrsc; and, ii) others identified by phage display in E. coli (Mukhija and Berni, 1997; Mukhija et al., 2008). Our results revealed that, for most of the peptides, the affinities were in the range of 15 ¿M, being slightly larger in those which involved HPr- and phage-derived peptides (KD ~5 ¿M). Since the affinity of EINsc for HPrsc is 12 ¿M, we suggest that all the assayed peptides can be considered as good target compounds to inhibit the interaction between HPr and EIN.


Fundación Dialnet

Dialnet Plus

  • Más información sobre Dialnet Plus

Opciones de compartir

Opciones de entorno