Heterocyclic Hemipiperazines: Multistimuli-responsive Switches and Sensors for Zinc or Cadmium Ions.

Advance in the design of molecular photoswitches - adapters that convert light into changes at molecular level - opens up exciting possibilities in preparing smart polymers, drugs photoactivated inside humans, or light-fueled nanomachines that might in the future operate in our bloodstream.  Hemipiperazines are recently reported biocompatible molecular photoswitches based on cyclic dipeptides. Here we report a multistimuli-responsive hemipiperazine-based switch that reacts on light, solvents, acidity, or metal ions. Its photoequilibration is controlled by the intramolecular hydrogen bonding pattern. The compound can be used as a mid-nanomolar photoswitchable fluorescent sensor for zinc and cadmium ions, applicable to monitor environmental pollution in real time.

Reversible Influence of Hemipiperazine Photochromism on the Early Development of Zebrafish Embryo.

This study explores the potential of controlling organismal development with light by using reversible photomodulation of activity in bioactive compounds. Specifically, our research focuses on plinabulin 1, an inhibitor of tubulin dynamics that contains a photochromic motif called hemipiperazine. The two isomeric forms, Z-1 and E-1, can partially interconvert with light, yet show remarkable thermal stability in darkness. The Z-isomer exhibits higher cytotoxicity due to stronger binding to α-tubulin's colchicine site. The less toxic E-1 form, considered a "pro-drug", can be isolated in vitro and stored. Upon activation by blue or cyan light, it predominantly generates the more toxic Z-1 form. Here we demonstrate that 1 can effectively photomodulate epiboly, a critical microtubule-dependent cell movement during gastrulation in zebrafish embryos. This research highlights the potential of photomodulation for precise and reversible control of cellular activities and organismal development.

Molecular Syringe for Cargo Photorelease: Red-Light-Triggered Supramolecular Hydrogel.

Photochromic supramolecular hydrogels are versatile materials that show macroscopic effects upon irradiation, like liquefaction or shape changes. Here, we demonstrate a simple photochromic cyclic dipeptide (2,5-diketopiperazine-based) supergelator, composed of (S)-lysine and an azobenzene analogue of phenylalanine, that forms supramolecular hydrogels even at 0.1 wt% loading. The gels can physically encapsulate cargo molecules and release them to the environment in a controllable manner upon irradiation with red light, thus working as a "molecular syringe". As the material is biocompatible and operational in the "therapeutic window" of light (>650 nm) that deeply penetrates soft human tissues, it is applicable to smart drug-delivery systems.

Heterocyclic Hemipiperazines: Water-Compatible Peptide-Derived Photoswitches.

Hemipiperazines are a recently discovered class of peptide-derived molecular photoswitches with high biocompatibility and therapeutic potential. Here, for the first time we describe photochromism of heterocyclic hemipiperazines. They demonstrate long thermal lifetimes, and enlarged band separation between photoisomers. Efficient photoisomerization occurs under aqueous conditions, although with a need for organic co-solvent. Bidirectional switching with visible light is observed for an extended aromatic system.

Hemipiperazines as peptide-derived molecular photoswitches with low-nanomolar cytotoxicity.

Molecular photoswitches transform light energy into reversible structural changes. Their combination with known pharmacophores often allows for photomodulation of the biological activity. The effort to apply such compounds in photopharmacology as light-activated pro-drugs is, however, hampered by serious activity reduction upon pharmacophore modifications, or limited biostability. Here we report that a potent antimitotic agent plinabulin and its derivatives demonstrate up to 56-fold reversible activity photomodulation. Alternatively, irreversible photoactivation with cyan light can enhance the cytotoxicity up to three orders of magnitude-all without compromising the original activity level, as the original pharmacophore structure is unchanged. This occurs due to the presence of a peptide-derived photoswitchable motif hemipiperazine inside the plinabulin scaffold. Furthermore, we systematically describe photochromism of these thermally stable and biocompatible hemipiperazines, as well as a photoswitchable fluorophore derived from plinabulin. The latter may further expand the applicability of hemipiperazine photochromism towards super-resolution microscopy.

Photopharmacology of Antimitotic Agents.

Antimitotic agents such as the clinically approved vinca alkaloids, taxanes and epothilone can arrest cell growth during interphase and are therefore among the most important drugs available for treating cancer. These agents suppress microtubule dynamics and thus interfere with intracellular transport, inhibit cell proliferation and promote cell death. Because these drugs target biological processes that are essential to all cells, they face an additional challenge when compared to most other drug classes. General toxicity can limit the applicable dose and therefore reduce therapeutic benefits. Photopharmacology aims to avoid these side-effects by introducing compounds that can be applied globally to cells in their inactive form, then be selectively induced to bioactivity in targeted cells or tissue during a defined time window. This review discusses photoswitchable analogues of antimitotic agents that have been developed by combining different photoswitchable motifs with microtubule-stabilizing or microtubule-destabilizing agents.

Reversible photodissipation of composite photochromic azobenzene-alginate supramolecular hydrogels.

Supramolecular smart materials can quickly elicit macroscopic changes upon external stimulation. Here we report that an azobenzene-containing cyclic dipeptide can form composite supramolecular hydrogels with alginate based on the charge complementarity, at lower loading than the critical gelation concentrations of either component. The gels can reversibly dissipate to fluids with UV light. They can also encapsulate and photorelease fluorescent cargo. Upon treatment of the gels with aqueous calcium salts, the alginate component is permanently cross-linked and the photochromic component is solubilized.

Fluorinated Azobenzenes Switchable with Red Light.

Molecular photoswitches triggered with red or NIR light are optimal for photomodulation of complex biological systems, including efficient penetration of the human body for therapeutic purposes ("therapeutic window"). Yet, they are rarely reported, and even more rarely functional under aqueous conditions. In this work, fluorinated azobenzenes are shown to exhibit efficient E→Z photoisomerization with red light (PSS660nm >75 % Z) upon conjugation with unsaturated substituents. Initially demonstrated for aldehyde groups, this effect was also observed in a more complex structure by incorporating the chromophore into a cyclic dipeptide with propensity for self-assembly. Under physiological conditions, the latter molecule formed a supramolecular material that reversibly changed its viscosity upon irradiation with red light. Our observation can lead to design of new photopharmacology agents or phototriggered materials for in vivo use.

Recent Implementations of Molecular Photoswitches into Smart Materials and Biological Systems.

Light is a nearly ideal stimulus for molecular systems. It delivers information encoded in the form of wavelengths and their intensities with high precision in space and time. Light is a mild trigger that does not permanently contaminate targeted samples. Its energy can be reversibly transformed into molecular motion, polarity, or flexibility changes. This leads to sophisticated functions at the supramolecular and macroscopic levels, from light-triggered nanomaterials to photocontrol over biological systems. New methods and molecular adapters of light are reported almost daily. Recently reported applications of photoresponsive systems, particularly azobenzenes, spiropyrans, diarylethenes, and indigoids, for smart materials and photocontrol of biological setups are described herein with the aim to demonstrate that the 21st century has become the Age of Enlightenment-"Le siècle des Lumières"-in molecular sciences.

Evolution of a highly active and enantiospecific metalloenzyme from short peptides.

Primordial sequence signatures in modern proteins imply ancestral origins tracing back to simple peptides. Although short peptides seldom adopt unique folds, metal ions might have templated their assembly into higher-order structures in early evolution and imparted useful chemical reactivity. Recapitulating such a biogenetic scenario, we have combined design and laboratory evolution to transform a zinc-binding peptide into a globular enzyme capable of accelerating ester cleavage with exacting enantiospecificity and high catalytic efficiency (k cat/K M ~ 106 M-1 s-1). The simultaneous optimization of structure and function in a naïve peptide scaffold not only illustrates a plausible enzyme evolutionary pathway from the distant past to the present but also proffers exciting future opportunities for enzyme design and engineering.

Photocontrol of Drug Release from Supramolecular Hydrogels with Green Light.

Photoresponsive smart materials transform light energy into sophisticated functions. They find increasing biomedical applications in light-induced drug-release and photopharmacology, because they can provide the desired therapeutic effect locally due to precise spatiotemporal dosage control. However, the majority of reported studies rely on cytotoxic UV light that penetrates tissues poorly. Here, we report the first drug-releasing system based on photochromic low molecular weight supramolecular hydrogels that is triggered with visible light. We demonstrated green-light-induced release of structurally unmodified antibiotic, anticancer, and anti-inflammatory drugs under physiological conditions. Using the antibiotic-loaded gel, we selectively inhibited bacterial growth with green light.

Photoresponsive self-healing supramolecular hydrogels for light-induced release of DNA and doxorubicin.

An azobenzene-containing cyclic dipeptide PAP-DKP-Lys is a photoresponsive low-MW hydrogelator. The gelation process can be triggered with temperature, pH, light, and ionic strength. The resulting self-healing gels can encapsulate dsDNA or an anticancer drug doxorubicin, and release them in a light-dependent manner.

Exploration of alternate catalytic mechanisms and optimization strategies for retroaldolase design.

Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of ß-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.

Computational design of catalytic dyads and oxyanion holes for ester hydrolysis.

Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (k(cat)/K(M)) of 400 M(-1) s(-1) for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.

Efficient in vitro encapsulation of protein cargo by an engineered protein container.

An engineered variant of lumazine synthase, a nonviral capsid protein with a negatively charged luminal surface, is shown to encapsulate up to 100 positively supercharged green fluorescent protein (GFP) molecules in vitro. Packaging can be achieved starting either from intact, empty capsids or from capsid fragments by incubation with cargo in aqueous buffer. The yield of encapsulated GFP correlates directly with the host/guest mixing ratio, providing excellent control over packing density. Facile in vitro loading highlights the unusual structural dynamics of this novel nanocontainer and should facilitate diverse biotechnological and materials science applications.

Substrate screening identifies a novel target sequence for the proteasomal activity regulated by ionizing radiation.

The screening for treatment-induced enzyme activities offers the opportunity to discover important regulatory mechanisms and the identification of potential targets for anti-cancer therapies. A novel screening technique was applied to screen substrate peptide sequences for proteolytic activities up- or down-regulated by ionizing radiation in tumor cells. One specific substrate sequence was cleaved in control cell extracts but to a smaller extent in irradiated cell extracts and investigated in detail. Based on protease-class-specific inhibitory studies and cleavage site analysis a potent warhead-inhibitor was synthesized and used to identify the proteasome as the protease of interest. The investigated sequence shows high homology to a regulatory site of nucleoporin 50, an element of the nuclear pore complex, and site specific cleavage of nucleoporin 50 was determined in vitro suggesting a novel link between the ionizing radiation-regulated proteasome and nuclear protein shuttling.

Selection of bead-displayed, PNA-encoded chemicals.

The lack of efficient identification and isolation methods for specific molecular binders has fundamentally limited drug discovery. Here, we have developed a method to select peptide nucleic acid (PNA) encoded molecules with specific functional properties from combinatorially generated libraries. This method consists of three essential stages: (1) creation of a Lab-on-Bead library, a one-bead, one-sequence library that, in turn, displays a library of candidate molecules, (2) fluorescence microscopy-aided identification of single target-bound beads and the extraction--wet or dry--of these beads and their attached candidate molecules by a micropipette manipulator, and (3) identification of the target-binding candidate molecules via amplification and sequencing. This novel integration of techniques harnesses the sensitivity of DNA detection methods and the multiplexed and miniaturized nature of molecule screening to efficiently select and identify target-binding molecules from large nucleic acid encoded chemical libraries. Beyond its potential to accelerate assays currently used for the discovery of new drug candidates, its simple bead-based design allows for easy screening over a variety of prepared surfaces that can extend this technique's application to the discovery of diagnostic reagents and disease markers.

Imaging of mRNA in live cells using nucleic acid-templated reduction of azidorhodamine probes.

Nucleic acid-templated reactions leading to a fluorescent product represent an attractive strategy for the detection and imaging of cellular nucleic acids. Herein we report the use of a Staudinger reaction to promote the reduction of profluorescent azidorhodamine. The use of two cell-permeable GPNA probes, one labeled with the profluorescent azidorhodamine and the other with trialkylphosphine, enabled the detection of the mRNA encoding O-6-methylguanine-DNA methyltransferase in intact cells.

Nucleic acid encoding to program self-assembly in chemical biology.

This tutorial review serves as an introduction to the use of oligonucleotides and in particular peptide nucleic acids (PNAs) to encode function beyond heredity. Applications in chemical biology are reviewed starting with the use of nucleic acid tags to program self-assembled microarrays of small and macromolecules, followed by the use of nucleic acid templated reactions for the purpose of DNA or RNA sensing and finally, the use of nucleic acid templates to display ligands.

Fluorescence-based detection of single nucleotide permutation in DNA via catalytically templated reaction.

Templated reduction of low fluorescence azidocoumarin-PNA conjugate to high fluorescence aminocoumarin was achieved using a catalytic amount of DNA with single nucleotide resolution.