Self-assembled porphyrin-peptide cages for photodynamic therapy.

The development of photodynamic therapy requires access to smart photosensitizers which combine appropriate photophysical and biological properties. Interestingly, supramolecular and dynamic covalent chemistries have recently shown their ability to produce novel architectures and responsive systems through simple self-assembly approaches. Herein, we report the straightforward formation of porphyrin-peptide conjugates and cage compounds which feature on their surface chemical groups promoting cell uptake and specific organelle targeting. We show that they self-assemble, in aqueous media, into positively-charged nanoparticles which generate singlet oxygen upon green light irradiation, while also undergoing a chemically-controlled disassembly due to the presence of reversible covalent linkages. Finally, the biological evaluation in cells revealed that they act as effective photosensitizers and promote synergistic effects in combination with Doxorubicin.

Structure-Activity Relationships in Nucleic-Acid-Templated Vectors Based on Peptidic Dynamic Covalent Polymers.

The use of nucleic acids as templates, which can trigger the self-assembly of their own vectors represent an emerging, simple and versatile, approach toward the self-fabrication of tailored nucleic acids delivery vectors. However, the structure-activity relationships governing this complex templated self-assembly process that accompanies the complexation of nucleic acids remains poorly understood. Herein, the class of arginine-rich dynamic covalent polymers (DCPs) composed of different monomers varying the number and position of arginines were studied. The combinations that lead to nucleic acid complexation, in saline buffer, using different templates, from short siRNA to long DNA, are described. Finally, a successful peptidic DCP featuring six-arginine repeating unit that promote the safe and effective delivery of siRNA in live cancer cells was identified.

Fluorescent Dynamic Covalent Polymers for DNA Complexation and Templated Assembly.

Dynamic covalent polymers (DCPs) offer opportunities as adaptive materials of particular interest for targeting, sensing and delivery of biological molecules. In this view, combining cationic units and fluorescent units along DCP chains is attractive for achieving optical probes for the recognition and delivery of nucleic acids. Here, we report on the design of acylhydrazone-based DCPs combining cationic arginine units with π-conjugated fluorescent moieties based on thiophene-ethynyl-fluorene cores. Two types of fluorescent building blocks bearing neutral or cationic side groups on the fluorene moiety are considered in order to assess the role of the number of cationic units on complexation with DNA. The (chir)optical properties of the building blocks, the DCPs, and their complexes with several types of DNA are explored, providing details on the formation of supramolecular complexes and on their stability in aqueous solutions. The DNA-templated formation of DCPs is demonstrated, which provides new perspectives on the assembly of fluorescent DCP based on the nucleic acid structure.

Cell-Selective siRNA Delivery Using Glycosylated Dynamic Covalent Polymers Self-Assembled In†Situ by RNA Templating.

Dynamic covalent libraries enable exploring complex chemical systems from which bioactive assemblies can adaptively emerge through template effects. In this work, we studied dynamic covalent libraries made of complementary bifunctional cationic peptides, yielding a diversity of species from macrocycles to polymers. Although polymers are typically expressed only at high concentration, we found that siRNA acts as a template in the formation of dynamic covalent polymers at low concentration in a process guided by electrostatic binding. Using a glycosylated building block, we were able to show that this templated polymerization further translates into the multivalent presentation of carbohydrate ligands, which subsequently promotes cell uptake and even cell-selective siRNA delivery.

Combination of photodynamic therapy and gene silencing achieved through the hierarchical self-assembly of porphyrin-siRNA complexes.

In this work, we implemented a supramolecular approach in order to combine photodynamic therapy (PDT) with gene therapy. We made use of a simple cationic guanidylated porphyrin (H2­PG) with the hypothesis that porphyrin aggregates should be capable of complexing siRNA through multivalent interactions and thus contribute to its intracellular delivery, while remaining active photosensitizers for PDT. The PDT effect of H2­PG was shown by incubating human breast cancer cells (MDA-MB-231) with H2­PG followed by light-irradiation at 405 nm. On the other hand, while siRNA do not enter cells alone, we showed, by fluorescence confocal microscopy and flow cytometry, that H2­PG promotes the internalization of Atto-488 siRNA. Finally, studying the combined PDT and delivery of siRNA directed against inhibitory apoptotic protein (IAP) family, we found an additive effect of the two therapies, thereby demonstrating that H2­PG is capable of acting both as a photosensitizer and supramolecular siRNA vector.

Multivalent Metallosupramolecular Assemblies as Effective DNA Binding Agents.

We report the implementation of coordination chemistry onto the generation of new types of metallosupramolecular complexes with laterally appended cationic moieties for DNA binding in buffered aqueous media. Utilization of an N,N,O-type coordination pocket along with an octahedral zinc(II) metal ion allowed us to obtain mono- and tetranuclear complexes in both solution and solid state, as confirmed by NMR spectroscopy and single-crystal X-ray diffraction, respectively. By using isothermal titration calorimetry and gel electrophoresis, multiply charged cationic assemblies were observed to effectively bind to DNA through multivalent electrostatic interactions. Furthermore, we observed a correlation between the multivalency of the compounds employed and the effectiveness of DNA binding.

Biomolecular dynamic covalent polymers for DNA complexation and siRNA delivery.

Synthetic delivery systems that are described as smart are considered essential for the successful development of gene therapies. Dynamic covalent polymers (DCP) are dynamic and adaptive species that can expand and shorten their main chain in a reversible fashion. In particular, polyacylhydrazone DCPs are pH-sensitive and undergo hydrolytic dissociation at acidic pH, which is an interesting feature for gene delivery. Building upon our previous finding that cationic DCPs can complex DNA through multivalent interactions, we report here on a new generation of DCPs that incorporate modified amino acids. The covalent self-assembly through polycondensation was extended towards multifunctional DCPs combining different building blocks and different molecular dynamics. These biomolecular DCPs were found able to complex both long DNA and siRNA, and biological studies demonstrate that they are able to deliver functional siRNA in living cells. This straightforward and modular approach to the self-production of multifunctional and biomolecular DCPs as siRNA vectors can therefore constitute a stepping stone in smart gene delivery using dynamic and adaptive biodynamers.

Switching Multivalent DNA Complexation using Metal-Controlled Cationic Supramolecular Self-Assemblies.

We provide a proof-of-principle that coordination chemistry drives the in situ self-assembly of an inactive ligand into a multivalent cluster capable of effectively complexing DNA. We show that metal coordination and scavenging can be used to switch the multivalency of the system. Thus, controlled DNA complexation and decomplexation could be achieved.

One-Pot Self-Assembly of Peptide-Based Cage-Type Nanostructures Using Orthogonal Ligations.

The designed arrangement of biomolecular entities within monodisperse nanostructures is an important challenge toward functional biomaterials. We report herein a method for the formation of water-soluble peptide-based cages using orthogonal ligation reactions-acylhydrazone condensation and thiol-maleimide addition. The results show that using preorganized cyclic peptides and heterobifunctional spacers as building blocks and a set of orthogonal and chemoselective ligation reactions enable cage formation in one pot from six components and through eight reactions. Molecular modelling simulations reveal the structural dynamics of these structures. Finally, we exploited the reactional dynamics of the acylhydrazone by demonstrating the controlled dissociation of the cage through directed component exchange.

Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities.

Cyclophilins are peptidyl-prolyl cis/trans isomerases (PPIase) that catalyse the interconversion of the peptide bond at proline residues. Several cyclophilins play a pivotal role in the life cycle of a number of viruses. The existing cyclophilin inhibitors, all derived from cyclosporine A or sanglifehrin A, have disadvantages, including their size, potential for side effects unrelated to cyclophilin inhibition and drug-drug interactions, unclear antiviral spectrum and manufacturing issues. Here we use a fragment-based drug discovery approach using nucleic magnetic resonance, X-ray crystallography and structure-based compound optimization to generate a new family of non-peptidic, small-molecule cyclophilin inhibitors with potent in vitro PPIase inhibitory activity and antiviral activity against hepatitis C virus, human immunodeficiency virus and coronaviruses. This family of compounds has the potential for broad-spectrum, high-barrier-to-resistance treatment of viral infections.

Multivalent DNA recognition by self-assembled clusters: deciphering structural effects by fragments screening and evaluation as siRNA vectors.

The identification of low-molecular-weight clusters that effectively complex oligonucleotides of therapeutic interest is of great importance for applications in gene delivery. We recently reported the use of self-assembly processes based on chemoselective ligation in order to generate biomolecular clusters for the multivalent recognition of DNA. Herein, we exploit the modularity of this methodology to perform a one-pot fragments screening of scaffolds and binding groups. Structural parameters affecting DNA binding were observed and hits have been identified by fluorescence displacement and gel electrophoresis assays. Finally, we evaluated the potential of these systems for siRNA transfection. One biomolecular cluster was found to effectively complex and transport a 21-mer siRNA inside MCF7 human breast cancer cells, resulting in a significant knockdown of the target gene.

Dynamic Expression of DNA Complexation with Self-assembled Biomolecular Clusters.

We report herein the implementation of a dynamic covalent chemistry approach to the generation of multivalent clusters for DNA recognition. We show that biomolecular clusters can be expressed in situ by a programmed self-assembly process using chemoselective ligations. The cationic clusters are shown, by fluorescence displacement assay, gel electrophoresis and isothermal titration calorimetry, to effectively complex DNA through multivalent interactions. The reversibility of the ligation was exploited to demonstrate that template effects occur, whereby DNA imposes component selection in order to favor the most active DNA-binding clusters. Furthermore, we show that a chemical effector can be used to trigger DNA release through component exchange reactions.

Degradable hybrid materials based on cationic acylhydrazone dynamic covalent polymers promote DNA complexation through multivalent interactions.

The design of smart nonviral vectors for gene delivery is of prime importance for the successful implementation of gene therapies. In particular, degradable analogues of macromolecules represent promising targets as they would combine the multivalent presentation of multiple binding units that is necessary for achieving effective complexation of therapeutic oligonucleotides with the controlled degradation of the vector that would in turn trigger drug release. Toward this end, we have designed and synthesized hybrid polyacylhydrazone-based dynamic materials that combine bis-functionalized cationic monomers with ethylene oxide containing monomers. Polymer formation was characterized by (1) H and DOSY NMR spectroscopy and was found to take place at high concentration, whereas macrocycles were predominantly formed at low concentration. HPLC monitoring of solutions of these materials in aqueous buffers at pH values ranging from 5.0 to 7.0 revealed their acid-catalyzed degradation. An ethidium bromide displacement assay and gel electrophoresis clearly demonstrated that, despite being dynamic, these materials are capable of effectively complexing dsDNA in aqueous buffer and biological serum at N/P ratios comparable to polyethyleneimine polymers. The self-assembly of dynamic covalent polymers through the incorporation of a reversible covalent bond within their main chain is therefore a promising strategy for generating degradable materials that are capable of establishing multivalent interactions and effectively complexing dsDNA in biological media.

Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity.

In vertebrates, smooth muscle cells (SMCs) can reversibly switch between contractile and proliferative phenotypes. This involves various molecular mechanisms to reactivate developmental signaling pathways and induce cell dedifferentiation. The protein RBPMS2 regulates early development and plasticity of digestive SMCs by inhibiting the bone morphogenetic protein pathway through its interaction with NOGGIN mRNA. RBPMS2 contains only one RNA recognition motif (RRM) while this motif is often repeated in tandem or associated with other functional domains in RRM-containing proteins. Herein, we show using an extensive combination of structure/function analyses that RBPMS2 homodimerizes through a particular sequence motif (D-x-K-x-R-E-L-Y-L-L-F: residues 39-51) located in its RRM domain. We also show that this specific motif is conserved among its homologs and paralogs in vertebrates and in its insect and worm orthologs (CPO and MEC-8, respectively) suggesting a conserved molecular mechanism of action. Inhibition of the dimerization process through targeting a conserved leucine inside of this motif abolishes the capacity of RBPMS2 to interact with the translational elongation eEF2 protein, to upregulate NOGGIN mRNA in vivo and to drive SMC dedifferentiation. Our study demonstrates that RBPMS2 possesses an RRM domain harboring both RNA-binding and protein-binding properties and that the newly identified RRM-homodimerization motif is crucial for the function of RBPMS2 at the cell and tissue levels.

The RYMV-encoded viral suppressor of RNA silencing P1 is a zinc-binding protein with redox-dependent flexibility.

Viral suppressors of RNA interference (VSRs) target host gene silencing pathways, thereby operating important roles in the viral cycle and in host cells, in which they counteract host innate immune responses. However, the molecular mechanisms of VSRs are poorly understood. We provide here biochemical and biophysical features of the dual suppressor/activator VSR P1 protein encoded by the rice yellow mottle virus. In silico analyses of P1 suggested common features with zinc finger proteins and native mass spectrometry unambiguously confirmed that recombinant P1 binds reversibly two zinc atoms, each with a different strength. Additionally, we demonstrate that the reaction of P1 with H2O2 leads to zinc release, disulfide bond formation, and protein oligomerization. A reversible protein modification by redox alterations has only been described for a limited number of zinc finger proteins and has never been reported for VSRs. Those reported here for P1 might be a general feature of Cys-rich VSRs and could be a key regulatory mechanism for the control of RNA silencing.

Structural insight into the Mycobacterium tuberculosis Rv0020c protein and its interaction with the PknB kinase.

The protein Rv0020c from Mycobacterium tuberculosis, also called FhaA, is one of the major substrates of the essential Ser/Thr protein kinase (STPK) PknB. The protein is composed of three domains and is phosphorylated on a unique site in its N terminus. We solved the solution structure of both N- and C-terminal domains and demonstrated that the approximately 300 amino acids of the intermediate domain are not folded. We present evidence that the FHA, a phosphospecific binding domain, of Rv0020c does not interact with the phosphorylated catalytic domains of PknB, but with the phosphorylated juxtamembrane domain that links the catalytic domain to the mycobacterial membrane. We also demonstrated that the degree and the pattern of phosphorylation of this juxtamembrane domain modulates the affinity of the substrate (Rv0020c) toward its kinase (PknB).

Direct interaction between the Rice yellow mottle virus (RYMV) VPg and the central domain of the rice eIF(iso)4G1 factor correlates with rice susceptibility and RYMV virulence.

The adaptation of Rice yellow mottle virus (RYMV) to recessive resistance mediated by the rymv1-2 allele has been reported as a model to study the emergence and evolution of virulent variants. The resistance and virulence factors have been identified as eukaryotic translation initiation factor eIF(iso)4G1 and viral genome-linked protein (VPg), respectively, but the molecular mechanisms involved in their interaction are still unknown. In this study, we demonstrated a direct interaction between RYMV VPg and the central domain of rice eIF(iso)4G1 both in vitro, using recombinant proteins, and in vivo, using a yeast two-hybrid assay. Insertion of the E309K mutation in eIF(iso)4G1, conferring resistance in planta, strongly diminished the interaction with avirulent VPg. The efficiency of the major virulence mutations at restoring the interaction with the resistance protein was assessed. Our results explain the prevalence of virulence mutations fixed during experimental evolution studies and are consistent with the respective viral RNA accumulation levels of avirulent and virulent isolates. Our results also explain the origin of the residual multiplication of wild-type isolates in rymv1-2-resistant plants and the role of genetic context in the poor adaptability of the S2/S3 strain. Finally, the strategies of RYMV and members of family Potyviridae to overcome recessive resistance were compared.

Unique features of the folding landscape of a repeat protein revealed by pressure perturbation.

The volumetric properties of proteins yield information about the changes in packing and hydration between various states along the folding reaction coordinate and are also intimately linked to the energetics and dynamics of these conformations. These volumetric characteristics can be accessed via pressure perturbation methods. In this work, we report high-pressure unfolding studies of the ankyrin domain of the Notch receptor (Nank1-7) using fluorescence, small-angle x-ray scattering, and Fourier transform infrared spectroscopy. Both equilibrium and pressure-jump kinetic fluorescence experiments were consistent with a simple two-state folding/unfolding transition under pressure, with a rather small volume change for unfolding compared to proteins of similar molecular weight. High-pressure fluorescence, Fourier transform infrared spectroscopy, and small-angle x-ray scattering measurements revealed that increasing urea over a very small range leads to a more expanded pressure unfolded state with a significant decrease in helical content. These observations underscore the conformational diversity of the unfolded-state basin. The temperature dependence of pressure-jump fluorescence relaxation measurements demonstrated that at low temperatures, the folding transition state ensemble (TSE) lies close in volume to the folded state, consistent with significant dehydration at the barrier. In contrast, the thermal expansivity of the TSE was found to be equivalent to that of the unfolded state, indicating that the interactions that constrain the folded-state thermal expansivity have not been established at the folding barrier. This behavior reveals a high degree of plasticity of the TSE of Nank1-7.

Combination of noncovalent mass spectrometry and traveling wave ion mobility spectrometry reveals sugar-induced conformational changes of central glycolytic genes repressor/DNA complex.

The central glycolytic genes repressor (CggR) is a 37 kDa transcriptional repressor protein which plays a key role in Bacillus subtilis glycolysis by regulating the transcription of the gapA operon. Fructose-1,6-bisphosphate (FBP), identified as the effector sugar, has been shown to abolish the binding cooperativity of CggR to its DNA target and to modify the conformational dynamics of the CggR/DNA complex. In the present study, noncovalent mass spectrometry (MS) was used to obtain deeper insights into FBP-dependent CggR/DNA interactions. The effect of FBP binding on CggR alone and on CggR/DNA complexes was examined using automated chip-based nanoelectrospray MS and traveling wave ion mobility mass spectrometry (IM-MS). Our results revealed that tetrameric CggR dissociates into dimers upon FBP binding. Moreover, FBP binding to CggR/DNA complexes triggers disruption of intermolecular protein/protein interactions within the complex, significantly modifying its conformation as evidenced by a 5% increase of its collision cross section. For the first time, the use of IM-MS is reported to probe ligand-induced conformational modifications of a protein/DNA complex with an emphasis on the comparison with solution-based techniques.

Intrinsic disorder in Viral Proteins Genome-Linked: experimental and predictive analyses.

BACKGROUND: VPgs are viral proteins linked to the 5' end of some viral genomes. Interactions between several VPgs and eukaryotic translation initiation factors eIF4Es are critical for plant infection. However, VPgs are not restricted to phytoviruses, being also involved in genome replication and protein translation of several animal viruses. To date, structural data are still limited to small picornaviral VPgs. Recently three phytoviral VPgs were shown to be natively unfolded proteins. RESULTS: In this paper, we report the bacterial expression, purification and biochemical characterization of two phytoviral VPgs, namely the VPgs of Rice yellow mottle virus (RYMV, genus Sobemovirus) and Lettuce mosaic virus (LMV, genus Potyvirus). Using far-UV circular dichroism and size exclusion chromatography, we show that RYMV and LMV VPgs are predominantly or partly unstructured in solution, respectively. Using several disorder predictors, we show that both proteins are predicted to possess disordered regions. We next extend theses results to 14 VPgs representative of the viral diversity. Disordered regions were predicted in all VPg sequences whatever the genus and the family. CONCLUSION: Based on these results, we propose that intrinsic disorder is a common feature of VPgs. The functional role of intrinsic disorder is discussed in light of the biological roles of VPgs.