Luminescent lanthanide metallopeptides for biomolecule sensing and cellular imaging.

Lanthanide ions display unique luminescent properties that make them particularly attractive for the development of bioprobes, including long-lived excited states that allow the implementation of time-gated experiments and the elimination of background fluorescence associated with biological media, as well as narrow emission bands in comparison with typical organic fluorophores, which allow ratiometric and multiplex assays. These luminescent complexes can be combined with peptide ligands to endow them with additional targeting, responsiveness, and selectivity, thus multiplying the opportunities for creative probe design. In this feature article we will present some of the main strategies that researchers have used to develop lanthanide metallopeptide probes for the detection of proteins and nucleic acids, as well as for monitoring enzymatic activity and cellular imaging.

De Novo Engineering of Pd-Metalloproteins and Their Use as Intracellular Catalysts.

The development of transition metal-based catalytic platforms that promote bioorthogonal reactions inside living cells remains a major challenge in chemical biology. This is particularly true for palladium-based catalysts, which are very powerful in organic synthesis but perform poorly in the cellular environment, mainly due to their rapid deactivation. We now demonstrate that grafting Pd(II) complexes into engineered ß-sheets of a model WW domain results in cell-compatible palladominiproteins that effectively catalyze depropargylation reactions inside HeLa cells. The concave shape of the WW domain ß-sheet proved particularly suitable for accommodating the metal center and protecting it from rapid deactivation in the cellular environment. A thorough NMR and computational study confirmed the formation of the metal-stapled peptides and allowed us to propose a three-dimensional structure for this novel metalloprotein motif.

A copper(ii) peptide helicate selectively cleaves DNA replication foci in mammalian cells.

The use of copper-based artificial nucleases as potential anticancer agents has been hampered by their poor selectivity in the oxidative DNA cleavage process. An alternative strategy to solve this problem is to design systems capable of selectively damaging noncanonical DNA structures that play crucial roles in the cell cycle. We designed an oligocationic CuII peptide helicate that selectively binds and cleaves DNA three-way junctions (3WJs) and induces oxidative DNA damage via a ROS-mediated pathway both in vitro and in cellulo, specifically at DNA replication foci of the cell nucleus, where this DNA structure is transiently generated. To our knowledge, this is the first example of a targeted chemical nuclease that can discriminate with high selectivity 3WJs from other forms of DNA both in vitro and in mammalian cells. Since the DNA replication process is deregulated in cancer cells, this approach may pave the way for the development of a new class of anticancer agents based on copper-based artificial nucleases.

Dimeric Metal-Salphen Complexes Which Target Multimeric G-Quadruplex DNA.

G-Quadruplex DNA structures have attracted increasing attention due to their biological roles and potential as targets for the development of new drugs. While most guanine-rich sequences in the genome have the potential to form monomeric G-quadruplexes, certain sequences have enough guanine-tracks to give rise to multimeric quadruplexes. One of these sequences is the human telomere where tandem repeats of TTAGGG can lead to the formation of two or more adjacent G-quadruplexes. Herein we report on the modular synthesis via click chemistry of dimeric metal-salphen complexes (with NiII and PtII) bridged by either polyether or peptide linkers. We show by circular dichroism (CD) spectroscopy that they generally have higher selectivity for dimeric vs monomeric G-quadruplexes. The emissive properties of the PtII-salphen dimeric complexes have been used to study their interactions with monomeric and dimeric G-quadruplexes in vitro as well as to study their cellular uptake and localization.

Selective recognition of A/T-rich DNA 3-way junctions with a three-fold symmetric tripeptide.

Non-canonical DNA structures, particularly 3-Way Junctions (3WJs) that are transiently formed during DNA replication, have recently emerged as promising chemotherapeutic targets. Here, we describe a new approach to target 3WJs that relies on the cooperative and sequence-selective recognition of A/T-rich duplex DNA branches by three AT-Hook peptides attached to a three-fold symmetric and fluorogenic 1,3,5-tristyrylbenzene core.

Controlling oncogenic KRAS signaling pathways with a Palladium-responsive peptide.

RAS oncoproteins are molecular switches associated with critical signaling pathways that regulate cell proliferation and differentiation. Mutations in the RAS family, mainly in the KRAS isoform, are responsible for some of the deadliest cancers, which has made this protein a major target in biomedical research. Here we demonstrate that a designed bis-histidine peptide derived from the αH helix of the cofactor SOS1 binds to KRAS with high affinity upon coordination to Pd(II). NMR spectroscopy and MD studies demonstrate that Pd(II) has a nucleating effect that facilitates the access to the bioactive α-helical conformation. The binding can be suppressed by an external metal chelator and recovered again by the addition of more Pd(II), making this system the first switchable KRAS binder, and demonstrates that folding-upon-binding mechanisms can operate in metal-nucleated peptides. In vitro experiments show that the metallopeptide can efficiently internalize into living cells and inhibit the MAPK kinase cascade.

Stereoselective Self-Assembly of DNA Binding Helicates Directed by the Viral ß-Annulus Trimeric Peptide Motif.

Combining coordination chemistry and peptide engineering offers extraordinary opportunities for developing novel molecular (supra)structures. Here, we demonstrate that the ß-annulus motif is capable of directing the stereoselective assembly of designed peptides containing 2,2'-bipyridine ligands into parallel three-stranded chiral peptide helicates, and that these helicates selectively bind with high affinity to three-way DNA junctions.

Dynamic Stereoselection of Peptide Helicates and Their Selective Labeling of DNA Replication Foci in Cells*.

Although largely overlooked in peptide engineering, coordination chemistry offers a new set of interactions that opens unexplored design opportunities for developing complex molecular structures. In this context, we report new artificial peptide ligands that fold into chiral helicates in the presence of labile metal ions such as FeII and CoII . Heterochiral ß-turn-promoting sequences encode the stereoselective folding of the peptide ligands and define the physicochemical properties of their corresponding metal complexes. Circular dichroism and NMR spectroscopy in combination with computational methods allowed us to identify and determine the structure of two isochiral ΛΛ-helicates, folded as topological isomers. Finally, in addition to the in-vitro characterization of their selective binding to DNA three-way junctions, cell-microscopy experiments demonstrated that a rhodamine-labeled FeII helicate was internalized and selectively stains DNA replication factories in functional cells.

Stimuli-Responsive DNA Binding by Synthetic Systems.

DNA is the molecule responsible for the storage and transmission of the genetic information in living organisms. The expression of this information is highly regulated. In eukaryotes, it is achieved mainly at the transcription level thanks to specialized proteins called transcription factors (TFs) that recognize specific DNA sequences, thereby promoting or inhibiting the transcription of particular genes. In many cases, TFs are present in the cell in an inactive form but become active in response to an external signal, which might modify their localization and DNA binding properties or modulate their interactions with the rest of the transcriptional machinery. As a result of the crucial role of TFs, the design of synthetic peptides or miniproteins that can emulate their DNA binding properties and eventually respond to external stimuli is of obvious interest. On the other hand, although the B-form double helix is the most common DNA secondary structure, it is not the only one with an essential biological function. Guanine quadruplexes (GQs) have received considerable attention due to their critical role in the regulation of gene expression, which is usually associated with a change in the GQ conformation. Thus, the development of GQ probes whose properties can be controlled using external signals is also of significant relevance.In this Account, we present a summary of the recent efforts toward the development of stimuli-responsive synthetic DNA binders with a particular emphasis on our own contributions. We first introduce the structure of B and GQ DNAs, and some of the main factors underlying their selective recognition. We then discuss some of the different approaches used for the design of stimulus-mediated DNA binders. We have organized our discussion according to whether the interaction takes place with duplex or guanine quadruplex DNAs, and each section is divided according to the nature of the stimulus (i.e., physical or chemical). Regarding physical stimuli, light (through the incorporation of photolabile protecting groups or photoisomerizable agents) is the most common input for the activation/deactivation of DNA binding events. With respect to chemical signals, the use of metals (through the incorporation of metal-coordinating groups in the DNA binding agent) has allowed the development of a wide range of stimuli-responsive DNA binders. More recently, redox-based systems have also been used to control DNA interactions.This Account ends with a "Conclusions and Outlook" section highlighting some of the general lessons that have been learned and future directions toward further advancing the field.

Assembly of a Ternary Metallopeptide Complex at Specific DNA Sites Mediated by an AT-Hook Adaptor.

The nickel(II)-mediated self-assembly of a multimeric DNA binder is described. The binder is composed of two metal-chelating peptides derived from a bZIP transcription factor (brHis2 ) and one short AT-hook domain equipped with two bipyridine ligands (HkBpy2 ). These peptides reversibly assemble in the presence of NiII ions at selected DNA sequences of 13 base pairs.

MitoBlue as a tool to analyze the mitochondria-lysosome communication.

MitoBlue is a fluorescent bisamidine that can be used to easily monitor the changes in mitochondrial degradation processes in different cells and cellular conditions. MitoBlue staining pattern is exceptional among mitochondrial dyes and recombinant fluorescent probes, allowing the dynamic study of mitochondrial recycling in a variety of situations in living cells. MitoBlue is a unique tool for the study of these processes that will allow the detailed characterization of communication between mitochondria and lysosomes.

Ditopic binuclear copper(II) complexes for DNA cleavage.

Herein we present the synthesis of two ligands containing two di(2-picolyl)amine (DPA) units linked by either a 1,1'-(pyridine-2,6-diyl)bis(3-ethylurea) (L1) or a 1,1'-(1,3-phenylene)bis(3-ethylurea) (L2) spacer. The corresponding binuclear CuII and ZnII complexes were prepared and isolated. The X-ray structures of the L1 ligand and the [Cu2L1Cl2]2+ complex evidence an unusual cis/trans conformation of one of the urea groups stabilized by an intramolecular hydrogen bond with the nitrogen atom of the pyridyl spacer. The CuII complexes form rather strong ternary complexes with phosphorylated anions. The [Cu2L1]4+ complex presents a rather high affinity for pyrophosphate (logK11 = 8.19 at pH 7, 25 °C), while [Cu2L2]4+ stands out because of its strong binding to AMP2- (logK11 = 9.3 at pH 7, 25 °C). The interaction of the CuII complexes with deoxyribonucleic acid from calf thymus (ct-DNA) was monitored using circular dichroism (CD) and luminescence spectroscopies. These studies revealed a quite strong interaction of the complexes with ct-DNA (Kb = (6.4 ± 0.7) × 103 for [Cu2L1]4+ and Kb = (6.3 ± 1.0) × 103 for [Cu2L2]4+). Competition experiments carried out in the presence of methyl green and BAPPA (N1,N3-Bis(4-amidinophenyl)propane-1,3-diamine) as major and minor groove competitors, respectively, confirm that the interaction of both complexes with DNA takes place through the minor groove, in agreement with docking studies. The [Cu2L2]4+ complex is quite efficient in promoting the cleavage of the double-stranded pUC19 plasmid DNA, by favoring the conversion of the supercoiled form to the nicked form following a hydrolytic mechanism.

Canonical DNA minor groove insertion of bisbenzamidine-Ru(ii) complexes with chiral selectivity.

We report the first Ru(ii) coordination compounds that interact with DNA through a canonical minor groove insertion mode and with selectivity for A/T rich sites. This was made possible by integrating a bis-benzamidine minor groove DNA-binding agent with a ruthenium(ii) complex. Importantly, one of the enantiomers (Δ-[Ru(bpy)2 b4bpy]2+, Δ-4Ru) shows a considerably higher DNA affinity than the parent organic ligand and the other enantiomer, particularly for the AATT sequence, while the other enantiomer preferentially targets long AAATTT sites with overall lower affinity. Finally, we demonstrate that the photophysical properties of these new binders can be exploited for DNA cleavage using visible light.

A chemical approach for the synthesis of the DNA-binding domain of the oncoprotein MYC.

We describe the first chemical synthesis of a functional mutant of the DNA binding domain of the oncoprotein MYC, using two alternative strategies which involve either one or two Native Chemical Ligations (NCLs). Both routes allowed the efficient synthesis of a miniprotein which is capable of heterodimerizing with MAX, and replicate the DNA binding of the native protein. The versatility of the reported synthetic approach enabled the straightforward preparation of MYC and Omomyc analogues, as well as fluorescently labeled derivatives.

Directed Self-Assembly of Trimeric DNA-Bindingchiral Miniprotein Helicates.

We propose that peptides are highly versatile platforms for the precise design of supramolecular metal architectures, and particularly, for the controlled assembly of helicates. In this context, we show that the bacteriophage T4 Fibritin foldon (T4Ff) can been engineered on its N-terminus with metal-chelating 2,2'-bipyridine units that stereoselectively assemble in the presence of Fe(II) into parallel, three-stranded peptide helicates with preferred helical orientation. Modeling studies support the proposed self-assembly and the stability of the final helicate. Furthermore, we show that these designed mini-metalloproteins selectively recognize three-way DNA junctions over double-stranded DNA.

Triphenylphosphonium cation: A valuable functional group for antimicrobial photodynamic therapy.

Light-mediated killing of pathogens by cationic photosensitisers is a promising antimicrobial approach that avoids the development of resistance inherent to the use of antimicrobials. In this study, we demonstrate that modification of different photosensitisers with the triphenylphosphonium cation yields derivatives with excellent photoantimicrobial activity against Gram-positive bacteria (ie, Staphylococcus aureus and Enterococcus faecalis). Thus, the triphenylphosphonium functional group should be considered for the development of photoantimicrobials for the selective killing of Gram-positive bacteria in the presence of Gram-negative species.

DNA-binding miniproteins based on zinc fingers. Assessment of the interaction using nanopores.

Obtaining artificial proteins that mimic the DNA binding properties of natural transcription factors could open new ways of manipulating gene expression at will. In this context it is particularly interesting to develop simple synthetic systems. Inspired by the modularity of natural transcription factors, we have designed synthetic miniproteins that combine the zinc finger module of the transcription factor GAGA and AT-hook peptide domains. These constructs are capable of binding to composite DNA sequences of up to 14 base pairs with high affinity and good selectivity. In particular, we have synthesized three different chimeras and characterized their DNA binding properties by electrophoresis and fluorescence anisotropy. We have also used, for the first time in the study of peptide-based DNA binders, nanopore force spectroscopy to obtain further data on the DNA interaction.

Selective G-quadruplex binding by oligoarginine-Ru(dppz) metallopeptides.

A set of Ru(ii) metallopeptides containing the dppz ligand has been synthesized using SPPS methods. Fluorescence titration studies show that those metallopeptides featuring an octaarginine tail display a large binding preference for DNA G-quadruplex structures over those lacking it, and also that the interplay between the octoarginine functionalization and the ancillary ligand in the complex has an essential role in the recognition process. Furthermore, the oligoarginine metallopeptides are also efficiently internalized, causing cell death with signs of apoptosis.

Metal-Dependent DNA Recognition and Cell Internalization of Designed, Basic Peptides.

A fragment of the DNA basic region (br) of the GCN4 bZIP transcription factor has been modified to include two His residues at designed i and i+4 positions of its N-terminus. The resulting monomeric peptide (brHis2) does not bind to its consensus target DNA site (5'-GTCAT-3'). However, addition of Pd(en)Cl2 (en, ethylenediamine) promotes a high-affinity interaction with exquisite selectivity for this sequence. The peptide-DNA complex is disassembled by addition of a slight excess of a palladium chelator, and the interaction can be reversibly switched multiple times by playing with controlled amounts of either the metal complex or the chelator. Importantly, while the peptide brHis2 fails to translocate across cell membranes on its own, addition of the palladium reagent induces an efficient cell internalization of this peptide. In short, we report (1) a designed, short peptide that displays highly selective, major groove DNA binding, (2) a reversible, metal-dependent DNA interaction, and (3) a metal-promoted cell internalization of this basic peptide.

Anion Recognition as a Supramolecular Switch of Cell Internalization.

The cell internalization of designed oligoarginine peptides equipped with six glutamic acid residues and an anionic pyranine at the N-terminus is triggered upon addition of a supramolecular host. This host binds specifically to the pyranine moiety, enabling the complex to traverse the cell membrane. Interestingly, none of the components, neither the host nor the guest, are able to cross the cell membrane on their own.