A Bis-Zn2+ -Pyridyl-Salen-Type Complex Conjugated to the ATP Aptamer: An ATPase-Mimicking Nucleoapzyme.

Catalytic nucleic acids consisting of a bis-Zn2+ -pyridyl-salen-type ([di-ZnII 3,5 bis(pyridinylimino) benzoic acid]) complex conjugated to the ATP aptamer act as ATPase-mimicking catalysts (nucleoapzymes). Direct linking of the Zn2+ complex to the 3'- or 5'-end of the aptamer (nucleoapzymes I and II) or its conjugation to the 3'- or 5'-end of the aptamer through bis-thymidine spacers (nucleoapzymes III and IV) provided a set of nucleoapzymes exhibiting variable catalytic activities. Whereas the separated bis-Zn2+ -pyridyl-salen-type catalyst and the ATP aptamer do not show any noticeable catalytic activity, the 3'-catalyst-modified nucleoapzyme (nucleoapzyme IV) and, specifically, the nucleoapzyme consisting of the catalyst linked to the 3'-position through the spacer (nucleoapzyme III) reveal enhanced catalytic features in relation to the analogous nucleoapzyme substituted at the 5'-position (kcat =4.37 and 6.88 min-1 , respectively). Evaluation of the binding properties of ATP to the different nucleoapzyme and complementary molecular dynamics simulations suggest that the distance separating the active site from the substrate linked to the aptamer binding site controls the catalytic activities of the different nucleoapzymes.

Targeting an Interaction Between Two Disordered Domains by Using a Designed Peptide.

Invited for the cover of this issue is the group of Assaf Friedler at the Hebrew University of Jerusalem. The image depicts the protein-protein interactions reported in this work. Read the full text of the article at 10.1002/chem.202000465.

Targeting an Interaction Between Two Disordered Domains by Using a Designed Peptide.

Intrinsically disordered regions in proteins (IDRs) mediate many disease-related protein-protein interactions. However, the unfolded character and continuous conformational changes of IDRs make them difficult to target for therapeutic purposes. Here, we show that a designed peptide based on the disordered p53 linker domain can be used to target a partner IDR from the anti-apoptotic iASPP protein, promoting apoptosis of cancer cells. The p53 linker forms a hairpin-like structure with its two termini in close proximity. We designed a peptide derived from the disordered termini without the hairpin, designated as p53 LinkTer. The LinkTer peptide binds the disordered RT loop of iASPP with the same affinity as the parent p53 linker peptide, and inhibits the p53-iASPP interaction in vitro. The LinkTer peptide shows increased stability to proteolysis, penetrates cancer cells, causes nuclei shrinkage, and compromises the viability of cells. We conclude that a designed peptide comprising only the IDR from a peptide sequence can serve as an improved inhibitor since it binds its target protein without the need for pre-folding, paving the way for therapeutic targeting of IDRs.

Controlling the Catalytic Functions of DNAzymes within Constitutional Dynamic Networks of DNA Nanostructures.

Mimicking complex cellular dynamic chemical networks being up-regulated or down-regulated by external triggers is one of the challenges in systems chemistry. Constitutional dynamic networks (CDNs), composed of exchangeable components that respond to environmental triggers by self-adaption, provide general means to mimic biosystems. We use the structural and functional information encoded in nucleic acid nanostructures to construct effector (input)-triggered constitutional dynamic networks that reveal adaptable catalytic properties. Specifically, CDNs composed of four exchangeable constituents, AA', BA', AB', and BB', are constructed. In the presence of an effector (input) that controls the stability of one of the constituents, the input-guided up-regulation or down-regulation of the CDN's constituents proceeds. As effectors we apply the fuel-strand stabilization of one of the CDN constituents by the formation of the T-A·T triplex structure, or by the K+-ion-induced stabilization of one of the CDN constituents, via the formation of a K+-ion-stabilized G-quadruplex. Energetic stabilization of one of the CDN constituents leads to a new dynamically adapted network composed of up-regulated and down-regulated constituents. By applying counter triggers to the effector units, e.g., an antifuel strand or 18-crown-6-ether, reconfiguration to the original CDNs is demonstrated. The performance of the CDNs is followed by the catalytic activities of the constituents and by complementary quantitative gel electrophoresis experiments. The orthogonal triggered and switchable operation of the CDNs is highlighted.

An anticancer Ti(IV) complex increases mitochondrial reactive oxygen species levels in relation with hypoxia and endoplasmic-reticulum stress: A distinct non DNA-related mechanism.

PhenolaTi is a promising Ti(IV) anticancer complex, with high stability and cytotoxicity, without notable toxic side-effects. Its cellular mechanism was proposed to relate to ER stress. Herein, we investigated the downstream effects of this mode of action in two cancer cell lines: ovarian carcinoma A2780 and cervical adenocarcinoma HeLa. First, although Ti(IV) is a non-redox metal, the formation of mitochondrial reactive oxygen species (ROS) was detected with live-cell imaging. Then, we inspected the effect of the mitochondrial ROS on cytotoxicity, using two methods: (a) addition of compounds that either elevate or reduce the mitochondrial glutathione concentration, thus affecting the oxidative state of the cells; and (b) scavenging mitochondrial ROS. Unlike the results observed for cisplatin, neither method influenced the cytotoxicity of phenolaTi, implying that ROS formation was a mere side effect of its activity. Additionally, live cell imaging displayed the hypoxia induced by phenolaTi, which can be associated with ROS formation. Overall, the results support the notion that ER-stress is the main cellular mechanism of phenolaTi, leading to hypoxia and mitochondrial ROS. The distinct mechanism of phenolaTi, which is different from that of cisplatin, combined with its stability and favorable anticancer properties, altogether make it a strong chemotherapeutic drug candidate.

Titanium complexes affect Bacillus subtilis biofilm formation.

Biofilms are surface or interface-associated communities of bacterial cells, embedded in a self-secreted extracellular matrix (ECM). Cells in biofilms are 100-1000 times more resistant to antibiotic treatment relative to planktonic cells due to various reasons, including the ECM acting as a diffusion barrier to antibiotic molecules, the presence of persister cells that divide slowly and are less susceptible to cell-wall targeting drugs, and the activation of efflux pumps in response to antibiotic stress. In this study we tested the effect of two titanium(iv) complexes that have been previously reported as potent and non-toxic anticancer chemotherapeutic agents on Bacillus subtilis cells in culture and in biofilm forming conditions. The Ti(iv) complexes tested, a hexacoordinate diaminobis(phenolato)-bis(alkoxo) complex (phenolaTi) and a bis(isopropoxo) complex of a diaminobis(phenolato) "salan"-type ligand (salanTi), did not affect the growth rate of cells in shaken cultures, however they did affect biofilm formation. Surprisingly, while phenolaTi inhibited biofilm formation, the presence of salanTi induced the formation of more mechanically robust biofilms. Optical microscopy images of biofilm samples in the absence and presence of Ti(iv) complexes suggest that Ti(iv) complexes affect cell-cell and/or cell-matrix adhesion, and that these are interfered with phenolaTi and enhanced by salanTi. Our results highlight the possible effect of Ti(iv) complexes on bacterial biofilms, which is gaining interest in light of the emerging relations between bacteria and cancerous tumors.

Binding of the anticancer Ti(IV) complex phenolaTi to serum proteins: Thermodynamic and kinetic aspects.

Titanium(IV) anticancer complexes are promising candidates for treatment of various cancers, and previous studies have pointed to possible interactions between Ti(IV) anticancer complexes and the serum proteins albumin and transferrin. Herein, we explored the binding of phenolaTi, a leading diaminobis(phenolato)bis(alkoxo) Ti(IV) anticancer complex, to serum proteins, and derived the binding constants and thermodynamic parameters. The results were compared with those obtained for a salan Ti(IV) bis(isopropoxo) complex and titanocene dichloride, studied under similar conditions. Human serum albumin (HSA) binds phenolaTi in a spontaneous, exothermic process, with a dissociation constant (Kd) of 47 ± 7 µM at room temperature. In the presence of transferrin, the Kd of phenolaTi increases by 2-fold, reflecting the competition between the two proteins over the complex, which was more dominant for the other, less hydrolytically stable complexes tested. Examining the kinetics of the binding, it reaches a maximum after ca. 6 h, and the bond partially dissociates after 24-36 h, presumably due to partial ligand hydrolysis in the absence of cells; nevertheless, the proteins HSA and transferrin have a negligible effect on cytotoxicity after 72 h of incubation, with a possible negative impact on cell entry at short incubation periods. Overall, HSA serves as a carrier for phenolaTi through both its known drug binding sites, presumably in its intact form, which is the species that actively penetrates the cells and inflects cytotoxicity.

Targeting Protein Interaction Hotspots Using Structured and Disordered Chimeric Peptide Inhibitors.

The main challenge in inhibiting protein-protein interactions (PPI) for therapeutic purposes is designing molecules that bind specifically to the interaction hotspots. Adding to the complexity, such hotspots can be within both structured and disordered interaction interfaces. To address this, we present a strategy for inhibiting the structured and disordered hotspots of interactions using chimeric peptides that contain both structured and disordered parts. The chimeric peptides we developed are comprised of a cyclic structured part and a disordered part, which target both disordered and structured hotspots. We demonstrate our approach by developing peptide inhibitors for the interactions of the antiapoptotic iASPP protein. First, we developed a structured, α-helical stapled peptide inhibitor, derived from the N-terminal domain of MDM2. The peptide bound two hotspots on iASPP at the low micromolar range and had a cytotoxic effect on A2780 cancer cells with a half-maximal inhibitory concentration (IC50) value of 10 ± 1 µM. We then developed chimeric peptides comprising the structured stapled helical peptide and the disordered p53-derived LinkTer peptide that we previously showed to inhibit iASPP by targeting its disordered RT loop. The chimeric peptide targeted both structured and disordered domains in iASPP with higher affinity compared to the individual structured and disordered peptides and caused cancer cell death. Our strategy overcomes the inherent difficulty in inhibiting the interactions of proteins that possess structured and disordered regions. It does so by using chimeric peptides derived from different interaction partners that together target a much wider interface covering both the structured and disordered domains. This paves the way for developing such inhibitors for therapeutic purposes.

Titanium Tackles the Endoplasmic Reticulum: A First Genomic Study on a Titanium Anticancer Metallodrug.

PhenolaTi is an advanced non-toxic anticancer chemotherapy; this inert bis(phenolato)bis(alkoxo) Ti(IV) complex demonstrates the intriguing combination of high and wide efficacy with no detected toxicity in animals. Here we unravel the cellular pathways involved in its mechanism of action by a first genome study on Ti(IV)-treated cells, using an attuned RNA sequencing-based available technology. First, phenolaTi induced apoptosis and cell-cycle arrest at the G2/M phase in MCF7 cells. Second, the transcriptome of the treated cells was analyzed, identifying alterations in pathways relating to protein translation, DNA damage, and mitochondrial eruption. Unlike for common metallodrugs, electrophoresis assay showed no inhibition of DNA polymerase activity. Reduced in vitro cytotoxicity with added endoplasmic reticulum (ER) stress inhibitor supported the ER as a putative cellular target. Altogether, this paper reveals a distinct ER-related mechanism by the Ti(IV) anticancer coordination complex, paving the way for wider applicability of related techniques in mechanistic analyses of metallodrugs.

Programmed DNAzyme-Triggered Dissolution of DNA-Based Hydrogels: Means for Controlled Release of Biocatalysts and for the Activation of Enzyme Cascades.

Acrylamide/acrylamide-modified nucleic acid copolymer chains provide building units for the construction of acrylamide-DNA hydrogels. Three different hydrogels are prepared by the cross-linking of the acrylamide-DNA chains with metal ion-dependent DNAzyme sequences and their substrates. The metal ion-dependent DNAzyme sequences used in the study include the Cu(2+)-, Mg(2+)-, and Zn(2+)-dependent DNAzymes. In the presence of the respective metal ions, the substrates of the respective DNAzymes are cleaved, leading to the separation of the cross-linking units and to the dissolution of the hydrogel. The different hydrogels were loaded with a fluorophore-modified dextran or with a fluorophore-functionalized glucose oxidase. Treatment of the different hydrogels with the respective ions led to the release of the loaded dextran or the enzyme, and the rates of releasing of the loaded macromolecules followed the order of Cu(2+) > Mg(2+) > Zn(2+). Also, the different hydrogels were loaded with the enzymes ß-galactosidase (ß-Gal), glucose oxidase (GOx), or horseradish peroxidase (HRP). In the presence of the appropriate metal ions, the respective hydrogels were dissolved, resulting in the activation of the ß-Gal/GOx or GOx/HRP bienzyme cascades and of the ß-Gal/GOx/HRP trienzyme cascade.