Wigner Distribution Deconvolution Adaptation for Live Ptychography Reconstruction.

We propose a modification of Wigner distribution deconvolution (WDD) to support live processing ptychography. Live processing allows to reconstruct and display the specimen transmission function gradually while diffraction patterns are acquired. For this purpose, we reformulate WDD and apply a dimensionality reduction technique that reduces memory consumption and increases processing speed. We show numerically that this approach maintains the reconstruction quality of specimen transfer functions as well as reduces computational complexity during acquisition processes. Although we only present the reconstruction for scanning transmission electron microscopy datasets, in general, the live processing algorithm we present in this paper can be applied to real-time ptychographic reconstruction for different fields of application.

Complex magnetism of B20-MnGe: from spin-spirals, hedgehogs to monopoles.

B20 compounds are the playground for various non-trivial magnetic textures such as skyrmions, which are topologically protected states. Recent measurements on B20-MnGe indicate no clear consensus on its magnetic behavior, which is characterized by the presence of either spin-spirals or three-dimensional objects interpreted to be a cubic lattice of hedgehogs and anti-hedgehogs. Utilizing a massively parallel linear scaling all-electron density functional algorithm, we find from full first-principles simulations on cells containing thousands of atoms that upon increase of the compound volume, the state with lowest energy switches across different magnetic phases: ferromagnetic, spin-spiral, hedgehog and monopole.

Molecular Mechanisms of Biased and Probe-Dependent Signaling at CXC-Motif Chemokine Receptor CXCR3 Induced by Negative Allosteric Modulators.

Our recent explorations of allosteric modulators with improved properties resulted in the identification of two biased negative allosteric modulators, BD103 (N-1-{[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimi-din2yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl}]butanamide) and BD064 (5-[(N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl-2-[4-fluoro-3-(trifluoromethyl)phenyl]acetamido)methyl]-2-fluorophenyl}boronic acid), that exhibited probe-dependent inhibition of CXC-motif chemokine receptor CXCR3 signaling. With the intention to elucidate the structural mechanisms underlying their selectivity and probe dependence, we used site-directed mutagenesis combined with homology modeling and docking to identify amino acids of CXCR3 that contribute to modulator binding, signaling, and transmission of cooperativity. With the use of allosteric radioligand RAMX3 ([3H]N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-[(1-methylpiperidin-4-yl)methyl]acetamide), we identified that F1313.32 and Y3087.43 contribute specifically to the binding pocket of BD064, whereas D1864.60 solely participates in the stabilization of binding conformation of BD103. The influence of mutations on the ability of negative allosteric modulators to inhibit chemokine-mediated activation (CXCL11 and CXCL10) was assessed with the bioluminescence resonance energy transfer-based cAMP and ß-arrestin recruitment assay. Obtained data revealed complex molecular mechanisms governing biased and probe-dependent signaling at CXCR3. In particular, F1313.32, S3047.39, and Y3087.43 emerged as key residues for the compounds to modulate the chemokine response. Notably, D1864.60, W2686.48, and S3047.39 turned out to play a role in signal pathway selectivity of CXCL10, as mutations of these residues led to a G protein-active but ß-arrestin-inactive conformation. These diverse effects of mutations suggest the existence of ligand- and pathway-specific receptor conformations and give new insights in the sophisticated signaling machinery between allosteric ligands, chemokines, and their receptors, which can provide a powerful platform for the development of new allosteric drugs with improved pharmacological properties.

Fluorescence- and Radiolabeling of [Lys4,Nle17,30]hPP Yields Molecular Tools for the NPY Y4 Receptor.

The neuropeptide Y (NPY) Y4 receptor (Y4R) is involved in energy homeostasis and considered a potential drug target for the treatment of obesity. Only a few molecular tools, i.e., radiolabeled and fluorescent ligands, for the investigation of the Y4R were reported. Previously, [Lys4]hPP proved to be an appropriate full-length PP analog to prepare a fluorescent ligand by derivatization at the ε-amino group. To preclude oxidation upon long-term storage, we replaced the two methionine residues in [Lys4]hPP by norleucine and prepared the corresponding [3H]propionylated ([3H]12) and cyanine labeled (13) peptides, which were characterized and compared with a set of reference compounds in binding (Y1, Y2, Y4, and Y5 receptors) and functional (luciferase gene reporter, beta-arrestin-1,2) Y4R assays. Both molecular probes proved to be useful in radiochemical and flow cytometric saturation and competition Y4R binding experiments. Most strikingly, there was a different influence of the composition of buffer on equilibrium binding and kinetics: [3H]12 affinity (Kd in Na+-free buffer: 1.1 nM) clearly decreased with increasing sodium ion concentration, whereas dissociation and Y4R-mediated internalization of 13 (Kd in Na+-free buffer: 10.8 nM) were strongly affected by the osmolarity of the buffer as demonstrated by confocal microscopy. Displacement of [3H]12 and 13 revealed a tendency to higher apparent affinities for a set of reference peptides in hypotonic (Na+-free) compared to isotonic buffers. The differences were negligible in the case of hPP but up to 270-fold in the case of GW1229 (GR231118). By contrast, no relevant influence of Na+ on Y5R affinity became obvious, when the radioligands [H]12 and [3H]propionyl-pNPY were investigated in saturation binding and competition binding.

Dimeric carbamoylguanidine-type histamine H2 receptor ligands: A new class of potent and selective agonists.

The bioisosteric replacement of the acylguanidine moieties in dimeric histamine H2 receptor (H2R) agonists by carbamoylguanidine groups resulted in compounds with retained potencies and intrinsic activities, but considerably improved stability against hydrolytic cleavage. These compounds achieved up to 2500 times the potency of histamine when studied in [(35)S]GTPγS assays on recombinant human and guinea pig H2R. Unlike 3-(imidazol-4-yl)propyl substituted carbamoylguanidines, the corresponding 2-amino-4-methylthiazoles revealed selectivity over histamine receptor subtypes H1R, H3R and H4R in radioligand competition binding studies. H2R binding studies with three fluorescent compounds and one tritium-labeled ligand, synthesized from a chain-branched precursor, failed due to pronounced cellular accumulation and high non-specific binding. However, the dimeric H2R agonists proved to be useful pharmacological tools for functional studies on native cells, as demonstrated for selected compounds by cAMP accumulation and inhibition of fMLP-stimulated generation of reactive oxygen species in human monocytes.

[(3) H]UR-DE257: development of a tritium-labeled squaramide-type selective histamine H2 receptor antagonist.

A series of new piperidinomethylphenoxypropylamine-type histamine H2 receptor (H2 R) antagonists with different substituted "urea equivalents" was synthesized and characterized in functional in vitro assays. Based on these data as selection criteria, radiosynthesis of N-[6-(3,4-dioxo-2-{3-[3-(piperidin-1-ylmethyl)phenoxy]propylamino}cyclobut-1-enylamino)hexyl]-(2,3-(3) H2 )propionic amide ([(3) H]UR-DE257) was performed. The radioligand (specific activity: 63 Ci mmol(-1) ) had high affinity for human, rat, and guinea pig H2 R (hH2 R, Sf9 cells: Kd , saturation binding: 31 nM, kinetic studies: 20 nM). UR-DE257 revealed high H2 R selectivity on membranes of Sf9 cells, expressing the respective hHx R subtype (Ki values: hH1 R: >10000 nM, hH2 R: 28 nM, hH3 R: 3800 nM, hH4 R: >10000 nM). In spite of insurmountable antagonism, probably due to rebinding of [(3) H]UR-DE257 to the H2 R (extended residence time), the title compound proved to be a valuable pharmacological tool for the determination of H2 R affinities in competition binding assays.

Synthesis and functional characterization of imbutamine analogs as histamine H3 and H4 receptor ligands.

Imbutamine (4-(1H-imidazol-4-yl)butanamine) is a potent histamine H3 (H3R) and H4 receptor (H4R) agonist (EC50 values: 3 and 66 nM, respectively). Aiming at improved selectivity for the H4R, the imidazole ring in imbutamine was methyl-substituted or replaced by various differently substituted heterocycles (1,2,3-triazoles, 1,2,4-triazoles, pyridines, pyrimidines) as potential bioisosteres. Investigations in [(35)S]GTPγS binding assays using membranes of Sf9 insect cells expressing the respective human histamine receptor subtype revealed only very weak activity of most of the synthesized hetarylalkylamines at both receptors. By contrast, the introduction of substituents at the 4-imidazolyl ring was most effective regarding H4R selectivity. This holds for methyl substitution in position 2 and, especially, in position 5. 5-Methylimbutamine (H4R: EC50 = 59 nM, α = 0.8) was equipotent with imbutamine at the hH4R, but revealed about 16-fold selectivity for the hH4R compared to the hH3R (EC50 980 nM, α = 0.36), whereas imbutamine preferred the hH3R. The functional activities were in agreement with radioligand binding data. The results support the hypothesis that, by analogy with histamine, methyl substitution in histamine homologs offers a way to shift the selectivity in favor of the H4R.

Synthesis and pharmacological characterization of new tetrahydrofuran based compounds as conformationally constrained histamine receptor ligands.

A series of tetrahydrofuran based compounds with a bicyclic core that provides conformational restriction were synthesized and investigated by radioligand displacement studies and functional [(35)S]GTPγS binding assays at the human histamine receptor (hHR) subtypes. The amines and ((1S,3R,5S,6R)- and ((1S,3S,5S,6R)-3-(1H-imidazol-5-yl)-2-oxabicyclo[3.1.0]hexan-6-yl)methanamine), exhibited submicromolar Ki values at the hH3R with 10-fold higher affinities than their corresponding (6S)-epimers and 25- and >34-fold selectivity over the hH4R, respectively. Both compounds act as neutral antagonists at the hH3R with KB values of 181 and 32 nM, respectively. The cyanoguanidines of the imidazole series and the oxazole analogues turned out to be inactive at all hHR subtypes.

[(3)H]UR-PLN196: a selective nonpeptide radioligand and insurmountable antagonist for the neuropeptide Y Y(2) receptor.

Radioing in on NPY: Attachment of a [2,3-(3)H]propionyl group through an appropriate linker to the guanidine group of an (S)-argininamide-type neuropeptide Y (NPY) Y(2) receptor antagonist resulted in a subtype-selective radioligand.

Reactivity assessment of chalcones by a kinetic thiol assay.

The electrophilic nature of chalcones (1,3-diphenylprop-2-en-1-ones) and many other α,ß-unsaturated carbonyl compounds is crucial for their biological activity, which is often based on thiol-mediated regulation processes. To better predict their biological activity a simple screening assay for the assessment of the second-order rate constants (k(2)) in thia-Michael additions was developed. Hence, a clear structure-activity relationship of 16 differentially decorated hydroxy-alkoxychalcones upon addition of cysteamine could be established. Moreover, amongst other naturally occurring α,ß-unsaturated carbonyl compounds k(2) values for curcumin and cinnamaldehyde were gained while cinnamic acids or esters gave no or very slow reactions.

Synthesis and application of the first radioligand targeting the allosteric binding pocket of chemokine receptor CXCR3.

Strategies for the identification of allosteric modulators of chemokine receptors largely rely on various cell-based functional assays. Radioligand binding assays are typically not available for allosteric binding sites. We synthesized, purified, and applied the first tritium-labeled allosteric modulator of the human chemokine receptor CXCR3 (RAMX3, [(3) H]N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-[(1-methylpiperidin-4-yl)methyl]acetamide). RAMX3 is chemically derived from 8-azaquinazolinone-type allosteric modulators and binds to the CXCR3 receptor with a K(d) value of 1.08 nM (specific activity: 80.4 Ci mmol(-1) ). Radioligand displacement assays showed potent negative cooperativity between RAMX3 and chemokine CXCL11, providing a basis for the use of RAMX3 to investigate other potential allosteric modulators. Additionally, the synthesis and characterization of a number of other full and truncated 8-azaquinazoline analogues were used to validate the binding properties of RAMX3. We demonstrate that RAMX3 can be efficiently used to facilitate the discovery and characterization of small molecules as allosteric modulators of the CXCR3 receptor.