Expanding the Toolbox of Photoswitches for DNA Nanotechnology Using Arylazopyrazoles.

Photoregulation is among the most promising tools for development of dynamic DNA nanosystems, due to its high spatiotemporal precision, biocompatibility, and ease of use. So far, azobenzene and its derivatives have shown high potential in photocontrolling DNA duplex hybridization by light-dependent photoisomerization. Despite many recent advances, obtaining sufficiently high photoswitching efficiency under conditions more suitable for work with DNA nanostructures are challenging. Here we introduce a pair of arylazopyrazoles as new photoswitches for efficient and reversible control of DNA hybridization achieved even at room temperature with a low number of required modifications. Their photophysical properties in the native state and in DNA strands result in near-quantitative isomerization rates by irradiation with UV and orange light. To demonstrate the applicability of these photoswitches, we have successfully applied one of them to open and close a DNA hairpin by light at room temperature.

Studying the Conformation of a Receptor Tyrosine Kinase in Solution by Inhibitor-Based Spin Labeling.

The synthesis of a spin label based on PD168393, a covalent inhibitor of a major anticancer drug target, the epidermal growth factor receptor (EGFR), is reported. The label facilitates the analysis of the EGFR structure in solution by pulsed electron paramagnetic resonance (EPR) spectroscopy. For various EGFR constructs, including near-full-length EGFR, we determined defined distance distributions between the two spin labels bound to the ATP binding sites of the EGFR dimer. The distances are in excellent agreement with an asymmetric dimer of the EGFR. Based on crystal structures, this dimer had previously been proposed to reflect the active conformation of the receptor but structural data demonstrating its existence in solution have been lacking. More generally, our study provides proof-of-concept that inhibitor-based spin labeling enables the convenient introduction of site-specific spin labels into kinases for which covalent or tight-binding small-molecule modulators are available.

In vitro activity of wALADin benzimidazoles against different life cycle stages of Plasmodium parasites.

wALADin1 benzimidazoles are specific inhibitors of δ-aminolevulinic acid dehydratase from Wolbachia endobacteria of filarial nematodes. We report that wALADin1 and two derivatives killed blood stage Plasmodium falciparum in vitro (50% inhibitory concentrations, 39, 7.7, and 12.8 µM, respectively). One of these derivatives inhibited gliding motility of Plasmodium berghei ANKA infectious sporozoites with nanomolar affinity and blocked invasion into hepatocytes but did not affect intrahepatocytic replication. Hence, wALADin1 benzimidazoles are tools to study gliding motility and potential antiplasmodial drug candidates.

wALADin benzimidazoles differentially modulate the function of porphobilinogen synthase orthologs.

The heme biosynthesis enzyme porphobilinogen synthase (PBGS) is a potential drug target in several human pathogens. wALADin1 benzimidazoles have emerged as species-selective PBGS inhibitors against Wolbachia endobacteria of filarial worms. In the present study, we have systematically tested wALADins against PBGS orthologs from bacteria, protozoa, metazoa, and plants to elucidate the inhibitory spectrum. However, the effect of wALADin1 on different PBGS orthologs was not limited to inhibition: several orthologs were stimulated by wALADin1; others remained unaffected. We demonstrate that wALADins allosterically modulate the PBGS homooligomeric equilibrium with inhibition mediated by favoring low-activity oligomers, while 5-aminolevulinic acid, Mg(2+), or K(+) stabilized high-activity oligomers. Pseudomonas aeruginosa PBGS could be inhibited or stimulated by wALADin1 depending on these factors and pH. We have defined the wALADin chemotypes responsible for either inhibition or stimulation, facilitating the design of tailored PBGS modulators for potential application as antimicrobial agents, herbicides, or drugs for porphyric disorders.

A selective inhibitor of heme biosynthesis in endosymbiotic bacteria elicits antifilarial activity in vitro.

Lymphatic filariasis and onchocerciasis are severe diseases caused by filarial worms and affect more than 150 million people worldwide. Endosymbiotic α-proteobacteria Wolbachia are essential for these parasites throughout their life cycle. Using a high-throughput chemical screen, we identified a benzimidazole compound, wALADin1, that selectively targets the δ-aminolevulinic acid dehydratase (ALAD) of Wolbachia (wALAD) and exhibits macrofilaricidal effects on Wolbachia-containing filarial worms in vitro. wALADin1 is a mixed competitive/noncompetitive inhibitor that interferes with the Mg(2+)-induced activation of wALAD. This mechanism inherently excludes activity against the Zn(2+)-dependent human ortholog and might be translatable to Mg(2+)-responsive orthologs of other bacterial or protozoan pathogens. The specificity profile of wALADin1 derivatives reveals chemical features responsible for inhibitory potency and species selectivity. Our findings validate wALADins as a basis for developing potent leads that meet current requirements for antifilarial drugs.

Anti-proliferative effect of cytohesin inhibition in gefitinib-resistant lung cancer cells.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib, have been proven to efficiently inhibit the proliferation of a subset of non small-cell lung cancers (NSCLC). Unfortunately, the majority of NSCLC expressing wild type EGFR is primarily resistant to EGFR-TKI treatment. Here, we show that the proliferation of the gefitinib-resistant NSCLC cell lines H460 and A549 is reduced by the small molecule SecinH3 which indirectly attenuates EGFR activation by inhibition of cytohesins, a class of recently discovered cytoplasmic EGFR activators. SecinH3 and gefitinib showed a synergistic antiproliferative effect, which correlated with a profound inhibition of Akt activation and survivin expression. Treating mice bearing H460 xenografts with SecinH3 showed the antiproliferative and pro-apoptotic effect of SecinH3 in vivo. Our data suggest that targeting the EGFR indirectly by inhibiting its cytoplasmic activators, the cytohesins, has the potential to improve the treatment of primarily EGFR-TKI resistant lung cancers.

A trifluoromethylphenyl diazirine-based SecinH3 photoaffinity probe.

The synthesis of a trifluoromethylphenyl diazirine photoaffinity probe of the cytohesin inhibitor SecinH3 is described. The probe exhibits improved labelling efficiency over a benzophenone-based probe and thus is more suitable for photoaffinity labelling in complex biological samples.

In trap fragmentation and optical characterization of rotaxanes.

The first experiments on trapped rotaxanes are presented, combining collision induced fragmentation and in-trap laser spectroscopy. The intrinsic optical properties of three rotaxanes and their non-interlocked building blocks (thread and macrocycle) isolated in a quadrupolar ion trap are investigated. The excitation and relaxation processes under thermal activation as well as under photo-activation are addressed. The light and collision induced fragmentation pathways show that the degradation mechanisms occurring in the rotaxane are highly dependent on the nature of the thread. In the prospective of operating photoswitchable molecules, photo-activation is achieved in a controlled way by depositing photo-energy in the desired sub-unit of a mechanically interlocked structure.

A double-stranded DNA rotaxane.

Mechanically interlocked molecules such as rotaxanes and catenanes have potential as components of molecular machinery. Rotaxanes consist of a dumb-bell-shaped molecule encircled by a macrocycle that can move unhindered along the axle, trapped by bulky stoppers. Previously, rotaxanes have been made from a variety of molecules, but not from DNA. Here, we report the design, assembly and characterization of rotaxanes in which both the dumb-bell-shaped molecule and the macrocycle are made of double-stranded DNA, and in which the axle of the dumb-bell is threaded through the macrocycle by base pairing. The assembly involves the formation of pseudorotaxanes, in which the macrocycle and the axle are locked together by hybridization. Ligation of stopper modules to the axle leads to the characteristic dumb-bell topology. When an oligonucleotide is added to release the macrocycle from the axle, the pseudorotaxanes are either converted to mechanically stable rotaxanes, or they disassemble by means of a slippage mechanism to yield a dumb-bell and a free macrocycle. Our DNA rotaxanes allow the fields of mechanically interlocked molecules and DNA nanotechnology to be combined, thus opening new possibilities for research into molecular machines and synthetic biology.

Conformational flexibility of a rotaxane thread probed by electronic spectroscopy in helium nanodroplets.

Ultrahigh-resolution spectroscopic studies have been performed to elucidate the conformational landscape of the succinamide-based thread 1 that is frequently employed in mechanically interlocked molecular assemblies. We show how dissolving single molecules into a helium nanodroplet enables us to resolve the broad absorption spectrum--which is normally observed--into the separate contributions of individual conformers that are populated under the employed experimental conditions. Excellent agreement is obtained with the results of molecular dynamics calculations. The absorption spectrum of each conformer reveals a splitting of the zero-phonon resonance that is different for each conformer and could thus serve as a spectral signature.

Stiff, and sticky in the right places: binding interactions in isolated mechanically interlocked molecules probed by mid-infrared spectroscopy.

We report the results of high-resolution spectroscopic studies on isolated, jet-cooled [2]rotaxanes. Employing IR absorption spectroscopy, we show how these noncovalently bound, multicomponent molecular systems that so far have been out of reach from high-resolution techniques, can now be characterized at an unprecedented level. IR absorption spectra of prototypical hydrogen-bond assembled rotaxanes as well as their associated threads and macrocycles are shown to provide a direct view on the effects of interlocking the macrocycle and thread, and to offer a straightforward approach for the study of their structural and dynamical properties.

Probing the structure of a rotaxane with two-dimensional infrared spectroscopy.

Femtosecond 2D-IR spectroscopy has been used to study the structure of a [2]rotaxane composed of a benzylic amide macrocycle that is mechanically interlocked onto a succinamide-based thread. Both the macrocycle and the thread contain carbonyl groups, and by determining the coupling between the stretching modes of these groups from the cross-peaks in the 2D-IR spectrum, the structure of the macrocycle-thread system has been probed. Our results demonstrate that 2D-IR spectroscopy can be used to observe structural changes in molecular devices on a picosecond time scale.

"Magic rod" rotaxanes: the hydrogen bond-directed synthesis of molecular shuttles under thermodynamic control.

[reaction: see text] Peptide [2]- and [3]rotaxanes are assembled in high yields under thermodynamic control using hydrogen bonding interactions and reversible cross olefin metathesis.