Nanostructures based on ammonium-terminated amphiphilic Janus dendrimers as camptothecin carriers with antiviral activity

The self-assembly in water of amphiphilic Janus dendrimers leads to micelles suitable to encapsulate camptothecin and act as therapy against the hepatitis C virus, HCV. Dendrimers consist of bis-MPA dendrons linked by their focal point through a CuAAC reaction. The hydrophilic dendron wears ammonium groups in its periphery whereas the lipophilic dendrons contain stearic acid chains. The morphology of the aggregates is controlled through the chemical structure of the dendrimers, particularly their molecular weight and the adequate combination of the lipophilic and hydrophilic blocks. Camptothecin-loaded dendrimer aggregates constitute effective systems to inhibit HCV replication and show low toxicity working at low drug concentrations.This work was financially supported by the MINECO-FEDER funds (under the projects CTQ2015-70174-P, MAT2015-66208-C3-1-P and BFU2013-47064-P), Miguel Servet Program from the Instituto de la Salud Carlos III (CP07/00289, CPII13/00017), Fondo de Investigaciones Sanitarias (PI15/00663) and the Gobierno de Aragón-FSE (E04, B89 and B01 research groups). A.L. and R.C-G thank the MECD for their FPU grants (FPU12/05210 and FPU13/3870). R.C-G thanks the DGA for the EPIF grant B136/13. The authors would like to acknowledge the use of Servicios Cientifico-Técnicos of CEQMA (Universidad de Zaragoza-CSIC), Servicio General de Apoyo a la Investigación-SAI (Universidad de Zaragoza), CIBA (IACS-Universidad de Zaragoza) and the LMA (INA-Universidad de Zaragoza).Peer reviewe

A look at ligand binding thermodynamics in drug discovery

Introduction: Drug discovery is a challenging endeavor requiring the interplay of many different research areas. Gathering information on ligand binding thermodynamics may help considerably in reducing the risk within a high uncertainty scenario, allowing early rejection of flawed compounds and pushing forward optimal candidates. In particular, the free energy, the enthalpy, and the entropy of binding provide fundamental information on the intermolecular forces driving such interaction. Areas covered: The authors review the current status and recent developments in the application of ligand binding thermodynamics in drug discovery. The thermodynamic binding profile (Gibbs energy, enthalpy, and entropy of binding) can be used for lead selection and optimization (binding enthalpy, selectivity, and adaptability). Expert opinion: Binding thermodynamics provides fundamental information on the forces driving the formation of the drug-target complex. It has been widely accepted that binding thermodynamics may be used as a decision criterion along the ligand optimization process in drug discovery and development. In particular, the binding enthalpy may be used as a guide when selecting and optimizing compounds over a set of potential candidates. However, this has been recently called into question by arguing certain difficulties and in the light of certain experimental examples.Peer Reviewe

Bis-MPA amphiphilic Janus dendrimers for camptothecin drug delivery

Resumen del trabajo presentado al V Encuentro sobre Dendrímeros, celebtrado en Zaragoza (España) del 25 al 26 de enero de 2016.Janus dendrimers are composed of two dendritic blocks offering different and specific terminal groups. It is then possible to combine distinctive properties in one unique molecule in order to provoke a synergetic or an antagonist effect. Therefore, amphiphilic Janus dendrimers were synthesized combining a lipophilic dendron with a hydrophilic dendron. Such molecules can self-assemble in water. Among all the dendrimers, polyesters of 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) are widely used in drug delivery as they are easy to synthesize, biocompatible and biodegradable. Camptothecin is a poorly water soluble antiviral cytotoxic drug sensible to water. The encapsulation of the drug inside amphiphilic Janus dendrimers would allow to increase the solubility of the drug in water, to protect it from hydrolysis and to reduce its cytotoxic side effect. Bis-MPA dendrons from 1st to 3rd generation were funcionalized at their periphery with glycine to form the hydrophilic block and with stearic acid (C17 chains) to form the lipophilic block. Afterwards, they were linked together with CuAAC to form a series of 4 amphiphilic Janus dendrimers with different Hydrophilic/Lipophilic Balances (HLB) and with different molecular weights. The Critical Aggregation Concentration in water was determined and the aggregates were studied by DLS and observed by TEM. They are biocompatible and do not present any antiviral activity. The influence of the size of the dendrimers and the HLB in the camptothecin encapsulation and the drug activity was studied by measuring the cytotoxicity, the antiviral activity and the release of the encapsulated drug. When HLB is too high, drug encapsulation is limited, and bigger lipophilic dendrons restrict the diffusion of the drug inside and outside the aggregates. Accordingly, three of the four dendrimers can effectively encapsulate the camptothecin using the solvent diffusion technique. This reduces camptothecin toxicity while its antiviral activity is maintained.Peer reviewe

In Vitro Antiviral Activity of Tyrosinase from Mushroom Agaricus bisporus against Hepatitis C Virus

Tyrosinases from a commercial Agaricus bisporus protein extract and directly isolated from white mushrooms were purified in order to obtaining the well-known tyrosinase from A. bisporus (TyrAB) of 45 kDa and a newly discovered 50 kDa tyrosinase isoform (Tyr50 kDa), and tested showing high antiviral activity against the hepatitis C virus for the first time. Cell toxicity and antiviral activity of tyrosinases were determined in cultured Huh 5-2 liver tumor cells transfected with a replicon system (a plasmid that includes all non-structural hepatitis C virus proteins and replicates autonomously). TyrAB was able to inhibit the replication of the hepatitis C virus without inducing toxicity in liver cells. In addition, the post-translational isoform Tyr50 kDa showed higher antiviral capacity than the former (up to 10 times greater), also exhibiting 10 times higher activity than the commercial drug Ribavirin®. This antiviral activity was directly proportional to the enzymatic activity of tyrosinases, as no antiviral capacity was observed in the inactive form of the enzymes. The tyrosinases approach could represent a new antiviral inhibition mechanism, through a plausible catalytic mechanism of selective hydroxylation of the key role of tyrosine residues in viral proteases

Unexpected thermodynamic signature for the interaction of hydroxymethylated DNA with MeCP2

Hydroxymethylated cytosine (5hmC) is a stable DNA epigenetic mark recognized by methyl-CpG binding protein 2 (MeCP2), which acts as a transcriptional regulator and a global chromatin-remodeling element. Because 5hmC triggers a gene regulation response markedly different from that produced by methylated cytosine (5mC), both modifications must affect DNA structure and/or DNA interaction with MeCP2 differently. MeCP2 is a six-domain intrinsically disordered protein (IDP) with two domains responsible for dsDNA binding: methyl-CpG binding domain (MBD) and intervening domain (ID). Here we report the detailed thermodynamic characterization of the interaction of hmCpG-DNA with MeCP2. We find that hmCpG-DNA interacts with MeCP2 in a distinctly different mode with a particular thermodynamic signature, compared to methylated or unmethylated DNA. In addition, we find evidence for Rett syndrome-associated mutations altering the interaction of MeCP2 with dsDNA in a cytosine modification-specific manner which may correlate with disease onset time and clinical severity score.This work was supported by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [BFU2016-78232-P to A.V.C., BES-2017-080739 to D.O.A.]; Ministry of Science and Innovation MCIN/AEI/10.13039/501100011033/ and “ERDF A way of Making Europe” [PID2021-127296OB-I00 to A.V.C.]; Miguel Servet Program from Instituto de Salud Carlos III [CPII13/00017 to OA]; Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, “Investing in your future”) [PI18/00349 and PI21/00394 to O.A.]; Diputación General de Aragón [Protein Targets and Bioactive Compounds Group E45_20R to A.V.C., and Digestive Pathology Group B25_20R to O.A.]; and the Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd)

Cationic poly(ester amide) dendrimers: alluring materials for biomedical applications

Novel cationic poly(ester amide) dendrimers have been synthesized by copper(i) azide-alkyne cycloaddition (CuAAC) of a tripropargylamine core and azide-terminated dendrons, in turn prepared by iterative amide coupling of the new monomer 2,2′-bis(glycyloxymethyl)propionic acid (bis-GMPA). The alternation of ester and amide groups provided a dendritic scaffold that was totally biocompatible and degradable in aqueous media at physiological and acidic pH. The tripodal dendrimers naturally formed rounded aggregates with a drug that exhibited low water solubility, camptothecin, thus improving its cell viability and anti-Hepatitis C virus (anti-HCV) activity. The presence of numerous peripheral cationic groups enabled these dendrimers to form dendriplexes with both pDNA and siRNA and they showed effective in vitro siRNA transfection in tumoral and non-tumoral cell lines.This work was financially supported by the MINECO-FEDER funds (under the projects CTQ2015-70174-P and MAT2015-66208-C3-1-P), the Miguel Servet Program from the Instituto de la Salud Carlos III (CPII13/00017 to O.A.), Instituto de Salud Carlos III and European Union (ERDF/ESF, ‘‘Investing in your future’’) (PI15/00663) and the Gobierno de Aragón-FSE (E04 and B01 research groups). Centro de Investigacion Biomédica en Red en Enfermedades Hepaticas y Digestivas (CIBERehd); and Asociación Española de Gastroenterologia (AEG). A. L. and R. C.-G. thank the MECD for their FPU grants (FPU12/05210 and FPU13/3870). R. G.-P. and R. C.-G. thank the DGA for their EPIF grants.Peer Reviewe

Differential NtcA responsiveness to 2-oxoglutarate underlies the diversity of C/N balance regulation in Prochlorococcus

Previous studies showed differences in the regulatory response to C/N balance in Prochlorococcus with respect to other cyanobacteria, but no information was available about its causes, or the ecological advantages conferred to thrive in oligotrophic environments. We addressed the changes in key enzymes (glutamine synthetase, isocitrate dehydrogenase) and the ntcA gene (the global nitrogen regulator) involved in C/N metabolism and its regulation, in three model Prochlorococcus strains: MED4, SS120, and MIT9313. We observed a remarkable level of diversity in their response to azaserine, a glutamate synthase inhibitor which increases the concentration of the key metabolite 2-oxoglutarate, used to sense the C/N balance by cyanobacteria. Besides, we studied the binding between the global nitrogen regulator (NtcA) and the promoter of the glnA gene in the same Prochlorococcus strains, and its dependence on the 2-oxoglutarate concentration, by using isothermal titration calorimetry, surface plasmon resonance, and electrophoretic mobility shift. Our results show a reduction in the responsiveness of NtcA to 2-oxoglutarate in Prochlorococcus, especially in the MED4 and SS120 strains. This suggests a trend to streamline the regulation of C/N metabolism in late-branching Prochlorococcus strains (MED4 and SS120), in adaptation to the rather stable conditions found in the oligotrophic ocean gyres where this microorganism is most abundant.Peer Reviewe

Shell cross-linked polymeric micelles as camptothecin nanocarriers for anti-HCV therapy

A suitable carrier for camptothecin to act as therapy against the hepatitis C virus is presented. The carrier relies on an amphiphilic hybrid dendritic-linear-dendritic block copolymer, derived from pluronic F127 and bis-MPA dendrons, that forms micelles in aqueous solution. The dendrons admit the incorporation of multiple photoreactive groups that allow the clean and effective preparation of covalently cross-linked polymeric micelles (CLPM), susceptible of loading hydrophilic and lipophilic molecules. Cell-uptake experiments using a newly designed fluorophore, derived from rhodamine B, demonstrate that the carrier favors the accumulation of its cargo within the cell. Furthermore, loaded with camptothecin, it is efficient in fighting against the hepatitis C virus while shows lower cytotoxicity than the free drug.This work was financially supported by the MINECO-FEDER funds (projects MAT2012-38538-CO3-01, CTQ2012-35692, and BFU2013-47064-P), Miguel Servet Program from Instituto de Salud Carlos III (CP07/00289, CPII13/00017), Fondo de Investigaciones Sanitarias (PI10/00186), and the Gobierno de Aragon-FSE (E04, B89 and B01 research groups). A. L. and R. C-G. thank the MEC for their FPU grants, FPU12/05210 and FPU13/3870, and R.GP and R. C.-G. thank the DGA for the EPIF grants, B054/12 and B136/13.Peer Reviewe

Stereoselective synthesis and biological evaluation as inhibitors of hepatitis C virus RNA polymerase of GSK3082 analogues with structural diversity at the 5-position

GSK3082 – a hepatitis C virus RNA polymerase inhibitor – and a series of analogues with structural diversity at the 5-position were prepared from a 2,2,4,5-tetrasubstituted pyrrolidine obtained with a well-defined stereochemistry from the 1,3-dipolar cycloaddition of the chiral imino ester derived from leucine tert-butyl ester and (R)-2,3-O-isopropylideneglyceraldehyde with methyl acrylate. The chiral 2,2-dimethyl-1,3-dioxolane moiety provided by the glyceraldehyde served as a synthetic equivalent for different substituents and functional groups and these transformations usually required mild reaction conditions and simple work-up procedures. The inhibitory activity of the resulting GSK3082 analogues was studied in vitro in a cell-based assay of the subgenomic HCV RNA replication system. Some of the analogues showed good inhibitory activity with IC50 values in the nanomolar concentration range.This work was financially supported by the Instituto de Salud Carlos III and European Union (ERDF/ESF, ‘Investing in your future’) (PI15/00663), Miguel Servet Program from the Instituto de la Salud Carlos III (CPII13/00017 to O.A.) and the Gobierno de Aragón-FSE (B01 research groups), Centro de Investigacion Biomédica en Red en Enfermedades Hepaticas y Digestivas (CIBERehd) and Asociación Española de Gastroenterologia (AEG) and grants FU2016-78232-P (MINECO, Spain) and E45_17R (Gobierno de Aragón, Spain). R.C-G thanks the MECD for an FPU grant (FPU13/3870). R.C-G thanks the DGA for an EPIF grant.Peer reviewe