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Combination therapy is becoming imperative for the treatment of many cancers, as it provides a higher chance of avoiding drug resistance and tumor recurrence. Among the resistance-conferring factors, the tumor microenvironment plays a major role, and therefore, represents a viable target for adjuvant therapeutic agents. Thus, hypoxia and extracellular acidosis are known to select for the most aggressive and resilient phenotypes and build poorly responsive regions of the tumor mass. Carbonic anhydrase (CA, EC 4.2.1.1) IX isoform is a surficial zinc metalloenzyme that is proven to play a central role in regulating intra and extracellular pH, as well as modulating invasion and metastasis processes. With its strong association and distribution in various tumor tissues and well-known druggability, this protein holds great promise as a target to pharmacologically interfere with the tumor microenvironment by using drug combination regimens. In the present review, we summarized recent publications revealing the potential of CA IX inhibitors to intensify cancer chemotherapy and overcome drug resistance in preclinical settings.

Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule F\"orster Resonance Energy Transfer (smFRET) is a powerful approach to inform on the structure and the dynamics of labeled molecules. If the dynamics occur on the sub-millisecond timescale, capturing and quantifying conformational dynamics can be challenging by intensity-based smFRET. Multiparameter fluorescence detection (MFD) addresses this challenge by simultaneously registering intensities and fluorescence lifetimes. Together, the mean donor fluorescence lifetime and the fluorescence intensities inform on the variance, and the mean FRET efficiency tells the conformational dynamics. Here, we present a general framework that relates average fluorescence lifetimes and intensities in smFRET counting histograms. Using this framework, we show how to compute parametric relations (FRET-lines) of these observables that facilitate a graphical interpretation of experimental data, can be used to test models, identify conformational states, resolve the connectivity of states, and can be applied to unstructured systems to infer properties of polymer chains or study fast protein folding. To simplify the graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data and show how to decouple the motion of the fluorescence labels from the conformational dynamics of the biomolecule., Comment: Main Text (47 pages, 14 figures, 3 tables) with Supporting Information (22 pages, 2 figures)

A substantial improvement of potency and selectivity of imidazoline-based inhibitors of hCA VII (a promising target for the treatment of seizures and neuropathic pain) was achieved by simply switching the position of the benzenesulfonamide moiety from N1 (as in the earlier reported series) to C2. Selectivity indices vs the off-target isoforms (hCA I, I, I and IV) greater than 100 were reached, which is exceedingly rare for hCA VII inhibitors. The drastic profile improvement of the new series has been rationalized by an additional hydrogen bonding with the nonconserved Q69 residue in the active site of hCA VII (absent in the other three isoforms studied), which also results in a favorable accommodation of the inhibitor’s lipophilic periphery in the nearby hydrophobic pocket. The robustness of the docking simulations was tested and confirmed by molecular dynamics simulations.

A diverse set of mono- and bis-sulfonamide was obtained via a direct, chemoselective sulfochlorination of readily available yet hitherto unexplored N-arylpyrazole template. Biochemical profiling of compounds thus obtained against a panel of human carbonic anhydrases (hCA I, hCA II, hCA IV and hCA VII) revealed a number of leads that are promising from the isoform selectivity prospective and exhibit potent inhibition profile (from nanomolar to micromolar range). The observed SAR trends have been rationalized by in silico docking of selected compounds into the active site of all four isoforms. The results reported in this paper clearly attest to the power of direct sulfochlorination as the means to create carbonic anhydrase focused sets in order to identify isoform selective inhibitors of closely related enzymes., Graphical Abstract

Three distinct series of isoxazole-based primary mono- and bis-sulfonamides have been synthesized via direct sulfochlorination, each of them delivering nanomolar inhibitors of human carbonic anhydrase. Certain pronounced SAR trends have been established and rationalized by in silico docking. These findings expand the structure-activity knowledge base for heterocycle-containing sulfonamide carbonic anhydrase inhibitors and further validate the power of direct electrophilic sulfochlorination as a means of introducing the pharmacophoric primary sulfonamide group into structurally diverse aromatic precursors.

We present a fast hardware photon correlator implemented in a field-programmable gate array (FPGA) combined with a compact confocal fluorescence setup. The correlator has two independent units with a time resolution of 4 ns while utilizing less than 15% of a low-end FPGA. The device directly accepts transistor-transistor logic (TTL) signals from two photon counting detectors and calculates two auto- or cross-correlation curves in real time. Test measurements demonstrate that the performance of our correlator is comparable with the current generation of commercial devices. The sensitivity of the optical setup is identical or even superior to current commercial devices. The FPGA design and the optical setup both allow for a straightforward extension to multi-color applications. This inexpensive and compact solution with a very good performance can serve as a versatile platform for uses in education, applied sciences, and basic research.

The group of Wolfgang Wintermeyer has previously performed experiments on a protein Ffh from the Signal Recognition Particle (SRP) (Buskiewicz et al., JMB 2005). Using multiple double cystein mutants and labelling with Bodipy-FL they measured the changes of anisotropy for single labeled and double labeled Ffh in presence and absence of 4.5 sRNA in bulk. From these measurements, the extracted distances were compared to three different existing crystal structures of Ffh (Keenan et al., Cell 1998; Rosendal et al., PNAS 2003). The distances were in agreement with A/A structure of T. aquaticus (Keenan et all., Cell 1998).We use two recently developed approaches to study dynamics of Homo-FRET samples at single molecule level: Anisotropy photon distribution analysis (aPDA) and filtered fluorescence correlation spectroscopy. Anisotropy PDA is an extension of existing method (Kalinin et al., JPC 2007), and allows us to separate different populations by differences in anisotropy. Filtered FCS is a new and powerful modification of the standard FCS approach. Using the information of the fluorescence lifetime and anisotropy decays, we filter the signal and weight the contribution of each single photon to the corresponding population of the probe. The weighted time dependent signal is then correlated like a standard FCS, allowing for a sub-microsecond resolution. As the result we obtain auto- and cross-correlation curves of which cross-correlation curve allows for to extract species selected interconversion rates.Applying both tools on the homo-FRET labeled Ffh will allow us to determine the conformational space of the protein and the corresponding conformational kinetic in a single molecule experiment free in solution.

RNA three-way junctions are important ribosomal structural motifs. They are also widely used as building blocks and functional components in nanotechnology applications. Forster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling in combination with molecular dynamics simulations was used to determine the structure of RNA three-way junction (3WJ) without bulges, RNA three-way junction with a small (two unpaired nucleotides) bulge and RNA three way junction with bigger (5 unpaired nucleotides) bulge. In total 81 Donor-Acceptor pairs were measured using single-molecule multi-parameter fluorescence detection (smMFD). This allows us to observe structural changes of the molecule induced by addition of bulge to the initial structure. Rigid body models for the major conformers were obtained by docking rigid double-stranded A-RNA helices explicitly taking into account dye position distributions. This is done many times (1000 iterations) with random starting conformations, yielding all local minima. Obtained models were then refined by all-atom MD simulations [1,2]. First results indicate the presence of two coaxially stacked helices for 3WJs with additional bulges at the junction, and absence of such stacking in case with no bulge. noteworthy the stacked helices are different for the 3WJs with two and five nucleotides in the bulge.Our studies showed that high precision FRET measurements are a valuable tool to complement the structural information obtained by X-ray crystallography or NMR spectroscopy as these techniques are limited in detecting minority conformers.References1. Sindbert S, et al. (2011) Accur ate distance determination of nucleic acids via Forster resonance energy transfer: implications of dye linker length and rigidity. J Am Chem Soc 133(8):2463-2480.2. Kalinin S,et al. (2012) A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nat. Methods in revision.

Holliday junctions are four-way DNA junctions that occur during genetic recombination. In previous studies it was demonstrated that in the presence of Mg2+ ions these junctions continually switch between two stacking conformations with rates strongly dependent on the ion concentration. In the present study we performed FRET studies using confocal single molecule detection with diffusing molecules or evanescent excitation of immobilized single molecules as well as ensemble fluorescence lifetime studies. By this means the full time range of fluorescence from ns to s could be accessed in order to capture all dynamic ranges. A complex Mg-dependent kinetics was found. The simplest model that describes the observed kinetics consists of four states, with only two distinct FRET levels. All methods and analysis techniques consistently reveal continuous interconversion also in absence of Mg2+ whereas binding of Mg2+ "locks" the junctions in the respective states. Our results shape a new view on structural properties of four-way DNA junctions, identifying a dynamic equilibrium instead of an accumulation of a single open structure at low Mg2+.

We use single molecule FRET and the completeness of Multiparameter Fluorescence Detection to study protein flexibility and dye rigidity from submicro- to millisecond timescales in native and non-native conditions of the bacteriophage T4 Lysozyme (T4L).T4L contains 164-residues and consists of two domains connected by a long alpha helix. The enzyme cleaves the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine of bacterial cell wall saccharides. From crystalographic and EPR measurement the native enzyme is believed to be in dynamic exchange between various “open” conformations. Most likely a set of hinges is responsible for the dynamic motion of the enzyme. However, the time scale of these hinges is still unknown. To accurately determine protein conformations from single molecule FRET, one needs to consider the dynamic behavior of donor and acceptor fluorophores. To test protein and dye mobility, we used single and double labeled T4L mutants with various fluorophore linkers using site specific mutants. The orthogonal system contains a ketone handle in the N-terminal domain for reaction with a hydroxylamine or hydrazide fluorophore, or a thiol group in the C-terminal domain for reaction with a thiol-specific fluorophore.Furthermore, the double mutant is ideal for site-specific attachment of the donor and acceptor fluorophores. Beside studies on native conformational dynamics we look into non native conformational transitions using chemical denaturants.In addition, we complement our experimental work by modelling the accesible volume (AV) required for each fluorophore using steric interaction with known crystalographic data. The AV model considers all available locations where the dye could be located without causing sterical clashes. This tool in combination with single molecule FRET allows us to quantitatively determine corresponding structural distances from experimental data.