Self-assembling dendrimer nanosystems for specific fluorine magnetic resonance imaging and effective theranostic treatment of tumors.

Fluorine magnetic resonance imaging (19F-MRI) is particularly promising for biomedical applications owing to the absence of fluorine in most biological systems. However, its use has been limited by the lack of safe and water-soluble imaging agents with high fluorine contents and suitable relaxation properties. We report innovative 19F-MRI agents based on supramolecular dendrimers self-assembled by an amphiphilic dendrimer composed of a hydrophobic alkyl chain and a hydrophilic dendron. Specifically, this amphiphilic dendrimer bears multiple negatively charged terminals with high fluorine content, which effectively prevented intra- and intermolecular aggregation of fluorinated entities via electrostatic repulsion. This permitted high fluorine nuclei mobility alongside good water solubility with favorable relaxation properties for use in 19F-MRI. Importantly, the self-assembling 19F-MRI agent was able to encapsulate the near-infrared fluorescence (NIRF) agent DiR and the anticancer drug paclitaxel for multimodal 19F-MRI and NIRF imaging of and theranostics for pancreatic cancer, a deadly disease for which there remains no adequate early detection method or efficacious treatment. The 19F-MRI and multimodal 19F-MRI and NIRF imaging studies on human pancreatic cancer xenografts in mice confirmed the capability of both imaging modalities to specifically image the tumors and demonstrated the efficacy of the theranostic agent in cancer treatment, largely outperforming the clinical anticancer drug paclitaxel. Consequently, these dendrimer nanosystems constitute promising 19F-MRI agents for effective cancer management. This study offers a broad avenue to the construction of 19F-MRI agents and theranostics, exploiting self-assembling supramolecular dendrimer chemistry.

Cargo-selective and adaptive delivery of nucleic acid therapeutics by bola-amphiphilic dendrimers.

Nucleic acid therapeutics are becoming an important drug modality, offering the unique opportunity to address "undruggable" targets, respond rapidly to evolving pathogens, and treat diseases at the gene level for precision medicine. However, nucleic acid therapeutics have poor bioavailability and are chemolabile and enzymolabile, imposing the need for delivery vectors. Dendrimers, by virtue of their well-defined structure and cooperative multivalence, represent precision delivery systems. We synthesized and studied bola-amphiphilic dendrimers for cargo-selective and on-demand delivery of DNA and small interfering RNA (siRNA), both important nucleic acid therapeutics. Remarkably, superior performances were achieved for siRNA delivery with the second-generation dendrimer, yet for DNA delivery with the third generation. We systematically studied these dendrimers with regard to cargo binding, cellular uptake, endosomal release, and in vivo delivery. Differences in size both of the dendrimers and their nucleic acid cargos impacted the cooperative multivalent interactions for cargo binding and release, leading to cargo-adaptive and selective delivery. Moreover, both dendrimers harnessed the advantages of lipid and polymer vectors, while offering nanotechnology-based tumor targeting and redox-responsive cargo release. Notably, they allowed tumor- and cancer cell-specific delivery of siRNA and DNA therapeutics for effective treatment in different cancer models, including aggressive and metastatic malignancies, outperforming the currently available vectors. This study provides avenues to engineer tailor-made vectors for nucleic acid delivery and precision medicine.

Bola-Amphiphilic Glycodendrimers: New Carbohydrate-Mimicking Scaffolds to Target Carbohydrate-Binding Proteins.

Dendrimers are appealing scaffolds for creating carbohydrate mimics with unique multivalent cooperativity. We report here novel bola-amphiphilic glycodendrimers bearing mannose and glucose terminals, and a hydrophobic thioacetal core responsive to reactive oxygen species. The peculiar bola-amphiphilic feature enabled stronger binding to lectin compared to conventional amphiphiles. In addition, these dendrimers are able to target mannose receptors and glucose transporters expressed at the surface of cells, thus allowing effective and specific cellular uptake. This highlights their great promise for targeted delivery.

Binding of the B-Raf Inhibitors Dabrafenib and Vemurafenib to Human Serum Albumin: A Biophysical and Molecular Simulation Study.

Drug binding to human serum albumin (HSA) significantly affects in vivo drug transport and biological activity. To gain insight into the binding mechanism of the two B-Raf tyrosine kinase inhibitors dabrafenib and vemurafenib to HSA, in this work, we adopted a combined strategy based on fluorescence spectroscopy, isothermal titration calorimetry (ITC), circular dichroism (CD), and molecular simulations. Both anticancer drugs are found to bind spontaneously and with a 1:1 stoichiometry within the same binding pocket, located in Sudlow's site II (subdomain IIIA) of the protein with comparable affinity and without substantially perturbing the protein secondary structure. However, the nature of each drug-protein interactions is distinct: whereas the formation of the dabrafenib/HSA complex is more entropically driven, the formation of the alternative vemurafenib/HSA assembly is prevalently enthalpic in nature. Kinetic analysis also indicates that the association rate is similar for the two drugs, whereas the residence time of vemurafenib within the HSA binding pocket is somewhat higher than that determined for the alternative B-Raf inhibitor.

Self-assembling supramolecular dendrimer nanosystem for PET imaging of tumors.

Bioimaging plays an important role in cancer diagnosis and treatment. However, imaging sensitivity and specificity still constitute key challenges. Nanotechnology-based imaging is particularly promising for overcoming these limitations because nanosized imaging agents can specifically home in on tumors via the "enhanced permeation and retention" (EPR) effect, thus resulting in enhanced imaging sensitivity and specificity. Here, we report an original nanosystem for positron emission tomography (PET) imaging based on an amphiphilic dendrimer, which bears multiple PET reporting units at the terminals. This dendrimer is able to self-assemble into small and uniform nanomicelles, which accumulate in tumors for effective PET imaging. Benefiting from the combined dendrimeric multivalence and EPR-mediated passive tumor targeting, this nanosystem demonstrates superior imaging sensitivity and specificity, with up to 14-fold increased PET signal ratios compared with the clinical gold reference 2-fluorodeoxyglucose ([18F]FDG). Most importantly, this dendrimer system can detect imaging-refractory low-glucose-uptake tumors that are otherwise undetectable using [18F]FDG. In addition, it is endowed with an excellent safety profile and favorable pharmacokinetics for PET imaging. Consequently, this dendrimer nanosystem constitutes an effective and promising approach for cancer imaging. Our study also demonstrates that nanotechnology based on self-assembling dendrimers provides a fresh perspective for biomedical imaging and cancer diagnosis.

Propellanes as Rigid Scaffolds for the Stereodefined Attachment of σ-Pharmacophoric Structural Elements to Achieve σ Affinity.

Following the concept of conformationally restriction of ligands to achieve high receptor affinity, we exploited the propellane system as rigid scaffold allowing the stereodefined attachment of various substituents. Three types of ligands were designed, synthesized and pharmacologically evaluated as σ1 receptor ligands. Propellanes with (1) a 2-methoxy-5-methylphenylcarbamate group at the "left" five-membered ring and various amino groups on the "right" side; (2) benzylamino or analogous amino moieties on the "right" side and various substituents at the left five-membered ring and (3) various urea derivatives at one five-membered ring were investigated. The benzylamino substituted carbamate syn,syn-4a showed the highest σ1 affinity within the group of four stereoisomers emphasizing the importance of the stereochemistry. The cyclohexylmethylamine 18 without further substituents at the propellane scaffold revealed unexpectedly high σ1 affinity (Ki = 34 nM) confirming the relevance of the bioisosteric replacement of the benzylamino moiety by the cyclohexylmethylamino moiety. Reduction of the distance between the basic amino moiety and the "left" hydrophobic region by incorporation of the amino moiety into the propellane scaffold resulted in azapropellanes with particular high σ1 affinity. As shown for the propellanamine 18, removal of the carbamate moiety increased the σ1 affinity of 9a (Ki = 17 nM) considerably. Replacement of the basic amino moiety by H-bond forming urea did not lead to potent σ ligands. According to molecular dynamics simulations, both azapropellanes anti-5 and 9a as well as propellane 18 adopt binding poses at the σ1 receptor, which result in energetic values correlating well with their different σ1 affinities. The affinity of the ligands is enthalpy driven. The additional interactions of the carbamate moiety of anti-5 with the σ1 receptor protein cannot compensate the suboptimal orientations of the rigid propellane and its N-benzyl moiety within the σ1 receptor-binding pocket, which explains the higher σ1 affinity of the unsubstituted azapropellane 9a.

Surface Charge of Supramolecular Nanosystems for In Vivo Biodistribution: A MicroSPECT/CT Imaging Study.

Bioimaging has revolutionized medicine by providing accurate information for disease diagnosis and treatment. Nanotechnology-based bioimaging is expected to further improve imaging sensitivity and specificity. In this context, supramolecular nanosystems based on self-assembly of amphiphilic dendrimers for single photon emission computed tomography (SPECT) bioimaging are developed. These dendrimers bear multiple In3+ radionuclides at their terminals as SPECT reporters. By replacing the macrocyclic 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid cage with the smaller 1,4,7-triazacyclononane-1,4,7-triacetic acid scaffold as the In3+ chelator, the corresponding dendrimer exhibits neutral In3+ -complex terminals in place of negatively charged In3+ -complex terminals. This negative-to-neutral surface charge alteration completely reverses the zeta-potential of the nanosystems from negative to positive. As a consequence, the resulting SPECT nanoprobe generates a highly sought-after biodistribution profile accompanied by a drastically reduced uptake in liver, leading to significantly improved tumor imaging. This finding contrasts with current literature reporting that positively charged nanoparticles have preferential accumulation in the liver. As such, this study provides new perspectives for improving the biodistribution of positively charged nanosystems for biomedical applications.

Direct Identification of α-Bisabolol Enantiomers in an Essential Oil Using a Combined Ion Mobility-Mass Spectrometry/Quantum Chemistry Approach.

Enantiomer-specific identification of chiral molecules in natural extracts is a challenging task, as many routine analytical techniques fail to provide selectivity in multicomponent mixtures. Here we describe an alternative approach, based on the combination of ion mobility-mass spectrometry (IM-MS) and quantum chemistry (QM), for the direct enantiomers differentiation in crude essential oils. The identification of α-bisabolol enantiomers contained in the raw essential oil (EO) from the Corsican Xanthium italicum fruits is reported as a proof-of-concept. Accordingly, IM-MS experiments performed in Ag+-doped methanol revealed the presence of both (+)- and (-)-α-bisabolol in the EO, while molecular simulations provided the structures of the two α-bisabolol enantiomer silver(I) adducts.

Mitochondrial dysfunction induced by a SH2 domain-targeting STAT3 inhibitor leads to metabolic synthetic lethality in cancer cells.

In addition to its canonical role in nuclear transcription, signal transducer and activator of transcription 3 (STAT3) is emerging as an important regulator of mitochondrial function. Here, we demonstrate that a novel inhibitor that binds with high affinity to the STAT3 SH2 domain triggers a complex cascade of events initiated by interference with mitochondrial STAT3 (mSTAT3). The mSTAT3-drug interaction leads to mitochondrial dysfunction, accumulation of proteotoxic STAT3 aggregates, and cell death. The cytotoxic effects depend directly on the drug's ability to interfere with mSTAT3 and mitochondrial function, as demonstrated by site-directed mutagenesis and use of STAT3 knockout and mitochondria-depleted cells. Importantly, the lethal consequences of mSTAT3 inhibition are enhanced by glucose starvation and by increased reliance of cancer cells and tumor-initiating cells on mitochondria, resulting in potent activity in cell cultures and tumor xenografts in mice. These findings can be exploited for eliciting synthetic lethality in metabolically stressed cancer cells using high-affinity STAT3 inhibitors. Thus, this study provides insights on the role of mSTAT3 in cancer cells and a conceptual framework for developing more effective cancer therapies.

Caution: Chemical Instability of Natural Biomolecules During Routine Analysis.

Natural products (NPs) constitute a significant source of active biomolecules widely used in medicine, pharmacology and cosmetics. However, NPs structural characterization has the drawback of their chemical instability during the extraction steps and their likely transformation during the analytical protocol. In particular, tamariscol and conocephalenol are two compounds largely used in the cosmetic industry for their odorant properties. Thus, in the present study, we focused on the evolution of these two metabolites (extracted from Frullania tamarisci and Conocephalum conicum, respectively), as followed by NMR. Interestingly, we found that, once dissolved in deuterated chloroform, these two tertiary alcohols are both subjected to transformation processes, leading to degradation compounds with altered structures. Accordingly, these detected degradation compounds have been fully characterized by NMR and the experimental findings were supported by computational chemistry data.

Perceptions and Misconceptions in Molecular Recognition: Key Factors in Self-Assembling Multivalent (SAMul) Ligands/Polyanions Selectivity.

Biology is dominated by polyanions (cell membranes, nucleic acids, and polysaccharides just to name a few), and achieving selective recognition between biological polyanions and synthetic systems currently constitutes a major challenge in many biomedical applications, nanovectors-assisted gene delivery being a prime example. This review work summarizes some of our recent efforts in this field; in particular, by using a combined experimental/computation approach, we investigated in detail some critical aspects in self-assembled nanomicelles and two major polyanions-DNA and heparin.

New flavonoid - N,N-dibenzyl(N-methyl)amine hybrids: Multi-target-directed agents for Alzheimer´s disease endowed with neurogenic properties.

The design of multi-target directed ligands (MTDLs) is a valid approach for obtaining effective drugs for complex pathologies. MTDLs that combine neuro-repair properties and block the first steps of neurotoxic cascades could be the so long wanted remedies to treat neurodegenerative diseases (NDs). By linking two privileged scaffolds with well-known activities in ND-targets, the flavonoid and the N,N-dibenzyl(N-methyl)amine (DBMA) fragments, new CNS-permeable flavonoid - DBMA hybrids (1-13) were obtained. They were subjected to biological evaluation in a battery of targets involved in Alzheimer's disease (AD) and other NDs, namely human cholinesterases (hAChE/hBuChE), ß-secretase (hBACE-1), monoamine oxidases (hMAO-A/B), lipoxygenase-5 (hLOX-5) and sigma receptors (σ1R/σ2R). After a funnel-type screening, 6,7-dimethoxychromone - DBMA (6) was highlighted due to its neurogenic properties and an interesting MTD-profile in hAChE, hLOX-5, hBACE-1 and σ1R. Molecular dynamic simulations showed the most relevant drug-protein interactions of hybrid 6, which could synergistically contribute to neuronal regeneration and block neurodegeneration.

A Dual Targeting Dendrimer-Mediated siRNA Delivery System for Effective Gene Silencing in Cancer Therapy.

Small interfering RNA (siRNA) is emerging as a novel therapeutic for treating various diseases, provided a safe and efficient delivery is available. In particular, specific delivery to target cells is critical for achieving high therapeutic efficacy while reducing toxicity. Amphiphilic dendrimers are emerging as novel promising carriers for siRNA delivery by virtue of the combined multivalent cooperativity of dendrimers with the self-assembling property of lipid vectors. Here, we report a ballistic approach for targeted siRNA delivery to cancer cells using an amphiphilic dendrimer equipped with a dual targeting peptide bearing an RGDK warhead. According to the molecular design, the amphiphilic dendrimer was expected to deliver siRNA effectively, while the aim of the targeting peptide was to home in on tumors via interaction of its warhead with integrin and the neuropilin-1 receptor on cancer cells. Coating the positively charged siRNA/dendrimer delivery complex with the negatively charged segment of the targeting peptide via electrostatic interactions led to small and stable nanoparticles which were able to protect siRNA from degradation while maintaining the accessibility of RGDK for targeting cancer cells and preserving the ability of the siRNA to escape from endosomes. The targeted system had enhanced siRNA delivery, stronger gene silencing, and more potent anticancer activity compared to nontargeted or covalent dendrimer-based systems. In addition, neither acute toxicity nor induced inflammation was observed. Consequently, this delivery system constitutes a promising nonviral vector for targeted delivery and can be further developed to provide RNAi-based personalized medicine against cancer. Our study also gives new perspectives on the use of nanotechnology based on self-assembling dendrimers in various biomedical applications.

Self-Assembled Nanomicelles as Curcumin Drug Delivery Vehicles: Impact on Solitary Fibrous Tumor Cell Protein Expression and Viability.

Solitary fibrous tumors (SFTs) are rare soft tissue sarcomas that rely on several epithelial-mesenchymal transition (EMT) protein regulators for invasion/metastatic progression. Curcumin (CUR) has several pharmacological activities, including anticancer activity and the ability to suppress the EMT process. However, poor absorption, rapid metabolism, and side effects at high doses limit the clinical applications of CUR. Here we present the results obtained by treating SFT cells with free CUR and three different CUR-loaded nanomicelles (NMs), each of which has its surface decorated with different ligands. All CUR-loaded NMs were more efficient in suppressing SFT cell viability and expression of EMT markers than CUR alone. Combined treatments with the pan-histone deacetylase dual inhibitor SAHA revealed a differential ability in inhibiting EMT markers expression and SFT cell invasiveness, depending on the NM-ligand type. Finally, combinations of photodynamic therapy and CUR-loaded NM administrations resulted in almost complete SFT cell viability abrogation 24 h after laser irradiation.

9-Aminoacridine-based agents impair the bovine viral diarrhea virus (BVDV) replication targeting the RNA-dependent RNA polymerase (RdRp).

Bovine viral diarrhea virus (BVDV) infection is still a plague that causes important livestock pandemics. Despite the availability of vaccines against BVDV, and the implementation of massive eradication or control programs, this virus still constitutes a serious agronomic burden. Therefore, the alternative approach to combat Pestivirus infections, based on the development of antiviral agents that specifically inhibit the replication of these viruses, is of preeminent actuality and importance. Capitalizing from a long-standing experience in antiviral drug design and development, in this work we present and characterize a series of small molecules based on the 9-aminoacridine scaffold that exhibit potent anti-BVDV activity coupled with low cytotoxicity. The relevant viral protein target - the RNA-dependent RNA polymerase - the binding mode, and the mechanism of action of these new antivirals have been determined by a combination of in vitro (i.e., enzymatic inhibition, isothermal titration calorimetry and site-directed mutagenesis assays) and computational experiments. The overall results obtained confirm that these acridine-based derivatives are promising compounds in the treatment of BVDV infections and, based on the reported structure-activity relationship, can be selected as a starting point for the design of a new generation of improved, safe and selective anti-BVDV agents.

Enantiomeric and Diastereomeric Self-Assembled Multivalent Nanostructures: Understanding the Effects of Chirality on Binding to Polyanionic Heparin and DNA.

A family of four self-assembling lipopeptides containing Ala-Lys peptides attached to a C16 aliphatic chain were synthesised. These compounds form two enantiomeric pairs that bear a diastereomeric relationship to one another (C16 -l-Ala-l-Lys/C16 -d-Ala-d-Lys) and (C16 -d-Ala-l-Lys/C16 -l-Ala-d-Lys). These diastereomeric pairs have very different critical micelle concentrations (CMCs). The self-assembled multivalent (SAMul) systems bind biological polyanions as a result of the cationic lysine groups on their surfaces. For heparin binding, there was no significant enantioselectivity, but there was a binding preference for the diastereomeric assemblies with lower CMCs. Conversely, for DNA binding, there was significant enantioselectivity for systems displaying d-lysine ligands, with a further slight preference for attachment to l-alanine, with the CMC being irrelevant.

Self-Assembled Multivalent (SAMul) Polyanion Binding-Impact of Hydrophobic Modifications in the Micellar Core on DNA and Heparin Binding at the Peripheral Cationic Ligands.

This paper reports a small family of cationic surfactants designed to bind polyanions such as DNA and heparin. Each molecule has the same hydrophilic cationic ligand and a hydrophobic aliphatic group with eighteen carbon atoms with one, two, or three alkene groups within the hydrophobic chain (C18-1, C18-2 and C18-3). Dynamic light scattering indicates that more alkenes lead to geometric distortion, giving rise to larger self-assembled multivalent (SAMul) nanostructures. Mallard Blue and Ethidium Bromide dye displacement assays demonstrate that heparin and DNA have markedly different binding preferences, with heparin binding most effectively to C18-1, and DNA to C18-3, even though the molecular structural differences of these SAMul systems are buried in the hydrophobic core. Multiscale modelling suggests that adaptive heparin maximises enthalpically favourable interactions with C18-1, while shape-persistent DNA forms a similar number of interactions with each ligand display, but with slightly less entropic cost for binding to C18-3-fundamental thermodynamic differences in SAMul binding of heparin or DNA. This study therefore provides unique insight into electrostatic molecular recognition between highly charged nanoscale surfaces in biologically relevant systems.

3D Homology Model of Sigma1 Receptor.

This chapter presents the three-dimensional (3D) model of the Sigma1 receptor protein as obtained from homology modeling techniques. We show the applicability of this structure to docking-based virtual screening and discuss combined in silico/in vitro mutagenesis studies performed to validate the structural features of the Sigma1 receptor model and to qualify/quantify the prominent role of specific amino acid residues in ligand binding. The validation of the virtual 3D Sigma1 receptor model and its reliable applicability to docking-based virtual screening is of significance for rational ligand design, even in light of the recently reported crystal structure for the Sigma1 receptor.

Fluorinated PET Tracers for Molecular Imaging of σ1 Receptors in the Central Nervous System.

At first the role of σ1 receptors in various neurological, psychiatric and neurodegenerative disorders is discussed. In the second part, the principle of positron emission tomography (PET ) is described and the known fluorinated PET tracers for labeling of σ1 receptors are presented. The third part focuses on fluoroalkyl substituted spirocyclic PET tracers, which represent the most promising class of fluorinated PET tracers reported so far. The homologous fluoroalkyl derivatives 12-15 show high σ1 affinity (K i = 0.59-1.4 nM) and high selectivity over the σ2 subtype (408-1331-fold). The enantiomers of the fluoroethyl derivative fluspidine 13 were prepared and pharmacologically characterized. Whereas the (S)-configured enantiomer (S)-13 (K i = 2.3 nM) is 4-fold less active than the (R)-enantiomer (R)-13 (K i = 0.57 nM), (S)-13 is metabolically more stable. The interactions of (S)-13 and (R)-13 with the σ1 receptor were analyzed at the molecular level using the 3D homology model. In an automated radiosynthesis [18F](S)-13 and [18F](R)-13 were prepared by nucleophilic substitution of the tosylates (S)-17 and (R)-17 with K[18F]F in high radiochemical yield, high radiochemical purity and short reaction time. Application of both enantiomers [18F](S)-13 and [18F](R)-13 to mice and piglets led to fast uptake into the brain, but [18F](R)-13 did not show washout from the brain indicating a quasi-irreversible binding. Both radiotracers [18F](S)-13 and [18F](R)-13 were able to label regions in the mouse and piglet brain with high σ1 receptor density. The specific binding of the enantiomeric tracers [18F](S)-13 and [18F](R)-13 could be replaced by the selective σ1 ligand SA4503.

Molecular dynamics reveal BCR-ABL1 polymutants as a unique mechanism of resistance to PAN-BCR-ABL1 kinase inhibitor therapy.

The acquisition of mutations within the BCR-ABL1 kinase domain is frequently associated with tyrosine kinase inhibitor (TKI) failure in chronic myeloid leukemia. Sensitive sequencing techniques have revealed a high prevalence of compound BCR-ABL1 mutations (polymutants) in patients failing TKI therapy. To investigate the molecular consequences of such complex mutant proteins with regards to TKI resistance, we determined by cloning techniques the presence of polymutants in a cohort of chronic-phase patients receiving imatinib followed by dasatinib therapy. The analysis revealed a high frequency of polymutant BCR-ABL1 alleles even after failure of frontline imatinib, and also the progressive exhaustion of the pool of unmutated BCR-ABL1 alleles over the course of sequential TKI therapy. Molecular dynamics analyses of the most frequent polymutants in complex with TKIs revealed the basis of TKI resistance. Modeling of BCR-ABL1 in complex with the potent pan-BCR-ABL1 TKI ponatinib highlighted potentially effective therapeutic strategies for patients carrying these recalcitrant and complex BCR-ABL1 mutant proteins while unveiling unique mechanisms of escape to ponatinib therapy.