Genetic and Neuroanatomical Support for Functional Brain Network Dynamics in Epilepsy

Focal epilepsy is a devastating neurological disorder that affects an overwhelming number of patients worldwide, many of whom prove resistant to medication. The efficacy of current innovative technologies for the treatment of these patients has been stalled by the lack of accurate and effective methods to fuse multimodal neuroimaging data to map anatomical targets driving seizure dynamics. Here we propose a parsimonious model that explains how large-scale anatomical networks and shared genetic constraints shape inter-regional communication in focal epilepsy. In extensive ECoG recordings acquired from a group of patients with medically refractory focal-onset epilepsy, we find that ictal and preictal functional brain network dynamics can be accurately predicted from features of brain anatomy and geometry, patterns of white matter connectivity, and constraints complicit in patterns of gene coexpression, all of which are conserved across healthy adult populations. Moreover, we uncover evidence that markers of non-conserved architecture, potentially driven by idiosyncratic pathology of single subjects, are most prevalent in high frequency ictal dynamics and low frequency preictal dynamics. Finally, we find that ictal dynamics are better predicted by white matter features and more poorly predicted by geometry and genetic constraints than preictal dynamics, suggesting that the functional brain network dynamics manifest in seizures rely on - and may directly propagate along - underlying white matter structure that is largely conserved across humans. Broadly, our work offers insights into the generic architectural principles of the human brain that impact seizure dynamics, and could be extended to further our understanding, models, and predictions of subject-level pathology and response to intervention

Noninvasive PET Imaging and Tracking of Engineered Human Muscle Precursor Cells for Skeletal Muscle Tissue Engineering

Transplantation of human muscle precursor cells (hMPCs) is envisioned for the treatment of various muscle diseases. However, a feasible noninvasive tool to monitor cell survival, migration, and integration into the host tissue is still missing. METHODS: In this study, we designed an adenoviral delivery system to genetically modify hMPCs to express a signaling-deficient form of human dopamine D2 receptor (hD2R). The gene expression levels of the receptor were evaluated by reverse transcriptase polymerase chain reaction, and infection efficiency was evaluated by fluorescent microscopy. The viability, proliferation, and differentiation capacity of the transduced cells, as well as their myogenic phenotype, were determined by flow cytometry analysis and fluorescent microscopy. (18)F-fallypride and (18)F-fluoromisonidazole, two well-established PET radioligands, were assessed for their potential to image engineered hMPCs in a mouse model and their uptakes were evaluated at different time points after cell inoculation in vivo. Biodistribution studies, autoradiography, and PET experiments were performed to determine the extent of signal specificity. To address feasibility for tracking hMPCs in an in vivo model, the safety of the adenoviral gene delivery was evaluated. Finally, the harvested tissues were histologically examined to determine whether survival of the transplanted cells was sustained at different time points. RESULTS: Adenoviral gene delivery was shown to be safe, with no detrimental effects on the primary human cells. The viability, proliferation, and differentiation capacity of the transduced cells were confirmed, and flow cytometry analysis and fluorescent microscopy showed that their myogenic phenotype was sustained. (18)F-fallypride and (18)F-fluoromisonidazole were successfully synthesized. Specific binding of (18)F-fallypride to hD2R hMPCs was demonstrated in vitro and in vivo. Furthermore, the (18)F-fluoromisonidazole signal was high at the early stages. Finally, sustained survival of the transplanted cells at different time points was confirmed histologically, with formation of muscle tissue at the site of injection. CONCLUSION: Our proposed use of a signaling-deficient hD2R as a potent reporter for in vivo hMPC PET tracking by (18)F-fallypride is a significant step toward potential noninvasive tracking of hD2R hMPCs and bioengineered muscle tissues in the clinic

Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M^(pro)), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M^(pro). In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2

Synthese, Evaluierung und 18 F-Markierung von neuen Liganden zur Visualisierung der Strychnin-insensitiven Bindungsstelle des NMDA-Rezeptors

Glutamat ist der wichtigste exzitatorische Neurotransmitter im Gehirn. Folglich spielen Glutamat-kontrollierte Rezeptorsysteme eine entscheidende Rolle in neurologischen Vorgängen, wie beispielsweise in Lern- und Gedächtnisprozessen. Gerade der NMDA-Rezeptor ist in eine Vielzahl solcher Vorgänge involviert und wird vor allem mit neurodegenerativen Erkrankungen wie Chorea Huntington, Morbus Alzheimer, Morbus Parkinson und zerebraler Ischämie in Verbindung gebracht. Folglich stellt die Visualisierung des NMDA-Rezeptorstatus eine Möglichkeit dar, den Verlauf solcher Prozesse zu untersuchen.rnDie Positronen- Emissions-Tomographie (PET) ist eine leistungsstarke Anwendung in der molekularen Bildgebung und erlaubt die in vivo-Visualisierung sowie Quantifizierung biochemischer Prozesse. Durch die Verwendung geeigneter Tracer können bestimmte pathologische und neurologische Abläufe beurteilt werden. rnZurzeit sind keine geeigneten PET-Tracer zur Untersuchung des NMDA-Rezeptors verfügbar. Bisher dargestellte PET-Liganden zeichneten sich durch nicht zufriedenstellende Affinitäten und Selektivitäten aus und führten meist auf Grund der hohen Lipophilie zu einem hohen Maß an unspezifischer Bindung. rnDie Strychnin-insensitive Glycinbindungsstelle des NMDA-Rezeptors stellt ein vielversprechendes Target dar, spezifische Liganden für diese Bindungsstelle zu synthetisieren. Hier zeichnen sich einige Verbindungsklassen durch exzellente Affinitäten und Selektivitäten sowie durch vielversprechende in vivo-Eigenschaften aus. rnAuf Grundlage dieser biologischen Daten wurden zwei Substanzen der 2-Indolcarbonsäure, nämlich die 4,6- Dichlor-3-(2-oxo-3-phenylimidazolidin-1-ylmethyl)-1H-indol-2-carbonsäure (MDJ-114) und die (E)-4,6-Dichlor-3-(2-phenylcarbamoylvinyl)-1H-indol-2-carbonsäure (GV150526), als Leitstruktur gewählt. Ferner wurde das 7-Chlor-4-hydroxy-3-(3-phenoxyphenyl)-1H-chinolin-2-on (L-701,324) aus der Substanzklasse der 4-Hydroxy-1H-chinolin-2-one als dritte Leitstruktur gewählt.rnFür diese Substanzen wurden 19F-markierte Analogverbindungen synthetisiert, um als inaktive Referenzverbindungen auf ihre Eignung überprüft zu werden. Hierzu wurde eine Fluorethoxygruppierung im terminalen Phenylring der entsprechenden Leitstruktur eingeführt. Durch Variation der Fluorethoxysubstitution in ortho-, meta- und para-Stellung, konnten die besten Affinitäten in einem kompetitiven Rezeptorbindungsassay durch Verdrängung von [3H]MDL-105,519 bestimmt werden. Als Maß für die Lipophilie wurden die entsprechenden log D-Werte über die HPLC-Methode bestimmt. Basierend auf den Ergebnissen der Evaluierung wurden zwei Derivate identifiziert, welche zur 18F-Markierung genutzt werden sollten (GV150526-Derivat 34: log D = 0,23 ± 0,03, IC50 = 0,20 ± 0,25 µM, Ki = 0,13 ± 0,16 µM; L701,324-Derivat 55: log D = - 0,25 ± 0,01, IC50 = 78 ± 37 µM, Ki = 51 ± 24 µM). Die 18F-Markierung erfolgte durch die Reaktion des entsprechenden Markierungsvorläufers mit dem Markierungssynthon 2-[18F]Fluorethyltosylat, welches durch die Umsetzung von Ethylenditosylat mit [18F]Fluorid hergestellt wurde. Die Radiosynthesen der beiden 18F-markierten Verbindungen [18F]34 (4,6-Dichlor-3-{2-[4-(2-[18F]fluorethoxy)-phenylcarbamoyl]-vinyl}-1H-indol-2-carbonsäure) und [18F]55 (7-Chlor-3-{3-[4-(2-[18F]fluorethoxy)-phenoxy]-phenyl}-4-hydroxy-1H-chinolin-2-on) wurden optimiert sowie semipräparative Abtrennverfahren entwickelt. Beide Tracer wurden auf ihre in vivo-Eignung im µPET-Experiment untersucht. Die Zeitaktivitätskurven lassen erkennen, dass beide Tracer entgegen der Erwartung nicht die Blut-Hirn-Schranke überwinden können. Für das GV150526-Derivat ([18F]34) wurden zusätzlich Autoradiographiestudien durchgeführt. Die erhaltenen Aufnahmen zeigten ein heterogenes Verteilungsmuster der Aktivitätsanreicherung. Ebenso wurde ein hohes Maß an unspezifischer Bindung beobachtet. Möglicherweise sind Cross-Affinitäten zu anderen Rezeptorsystemen oder der recht hohe lipophile Rest des Moleküls hierfür verantwortlich. Ein Grund für die unzureichende Hirngängigkeit der Radioliganden kann sich in der Carboxylatfunktion des GV150526-Derivats bzw. in der 4-Hydroxy-1H-chinolin-2-on-Einheit des L-701,324-Derivats wiederspiegeln. rnAuf Grundlage dieser Resultate können Versuche unternommen werden, für die Verbindungsklasse der 2-Indolcarbonsäuren entsprechende Ester als Prodrugs mit einer verbesserten Bioverfügbarkeit darzustellen. Ebenso können neue Strukturen als Grundlage für neue PET-Tracer untersucht werden.rnrnGlutamate controlled receptors play a central role in neuroexcitatory effects such as neuronal development and plasticity. Especially the NMDA receptor is involved in these neuronal functions. But also in case of neurodegenerative disorders like Parkinson’s disease, Alzheimers’s disease, cerebral ischemia and morbus Huntington, the NMDA receptor is of high interest. Therefore, the visualization of the NMDA receptor state is the option to study these processes.rnThe positron emission tomography (PET) is a powerful tool in molecular imaging sciences. Due to its high sensitivity it allows the in vivo visualization and quantification of biochemical processes and affords the investigation of pathological actions in neurosciences by the use of radiolabelled tracers. rnTo date, there are no suitable PET tracers, which can be used to study the NMDA receptor state. The loss of affinity and selectivity of synthesized PET ligands so far correlate with a high amount of unspecific binding. The glycin-binding site of the NMDA receptor is an appropriate target, to design novel tracers for the visualization of the receptor state. For this subtype, highly affine structures are known which show an excellent selectivity and in vivo activity.rnDue to these promising biological data, 4,6-dichloro-3-(2-oxo-3-phenylimidazolidin-1-ylmethyl)-1H-indole-2-carboxylic acid (MDJ-114) and (E)-4,6-dichloro-3-(2-phenylcarbamoylvinyl)-1H-indole-2-carboxylic acid (GV150526), both derivatives of the 3-imidazolidine-substitued 4,6-dichloro-indolecarboxylic acid, were dedicated for the design of PET analogues. In addition the 7-chloro-4-hydroxy-3-(3-phenoxyphenyl)-1H-quinolin-2-one (L-701,324) was chosen as an alternative compound with a different basic structure. rnBased on their promising biological data, these ligands were used to design novel derivatives for PET imaging. To determine their in vitro properties, the 19F-labelled analogues were synthesized by the introduction of a fluoroethoxy group in the terminal phenylring of the corresponding molecule. A variation of the fluoroethoxy position in ortho-, meta- and para-substitution allowed to determine the best affinity in a competitive receptor binding assay of [3H]MDL-105,519. To estimate the lipophily, log D-values were determined using the HPLC-method. Due to these data, the precursors of the GV150526- and L-701,324-derivatives were synthesized (34: log D = 0,23 ± 0,03, IC50 = 0,20 ± 0,25 µM, Ki = 0,13 ± 0,16 µM; 55: log D = - 0,25 ± 0,01, IC50 = 78 ± 37 µM, Ki = 51 ± 24 µM). These molecules were 18F-labelled by the use of the labelling agent 2-[18F]fluoro-ethyltosylate, which was generated by the reaction of ethylene ditosylate and [18F]fluoride to give the desired radiotracer [18F]34 (4,6-dichloro-3-{2-[4-(2-[18F]fluoroethoxy)-phenylcarbamoyl]-vinyl}-1H-indole-2-carboxylic acid) and [18F]55 (7-chloro-3-{3-[4-(2-[18F]fluoroethoxy)-phenoxy]-phenyl}-4-hydroxy-1H-quinolin-2-one). In both cases, the radiosynthesis was optimized to obtain the corresponding tracers in high radiochemical yields up to 86 %. For purification and quality control, HPLC methods were established.rnAdditionally for the indole derivative [18F]34, autoradiographic studies were performed. In Mg2+ and glutamate activated slices of the rat brain, [18F]34 showed a heterogeneous binding with no correspondence with the known distribution of NMDA-receptor rich areas. In addition, a high amount of unspecific binding was observed.rnBoth compounds were evaluated in µPET studies and showed low radioactivity levels in the rat brain. In contradiction with the findings of the unsubstituted derivatives both PET tracers are not suitable to penetrate the blood-brain-barrier and therefore cannot be used to image the NMDA receptor state in vivo. The carboxylate of the indole derivative and the 4-hydroxy-quinolin-2-one structure of thernL-701,324 derivative may be the main reason for this negative influence on blood-brain-barrier penetration. Due to these findings, new approaches can be started to synthesize prodrugs with improved bioavailability to investigate the NMDA receptor state. Additionally, new structures establish the possibility of the new design of novel potential PET tracers.r

closo-borane conjugated regulatory peptides retain high biological affinity: synthesis of closo-borane conjugated Tyr(3)-octreotate derivatives for BNCT

Despite the improvements in cancer therapy during the past years, high-grade gliomas and many other types of cancer are still extremely resistant to current forms of therapy. Boron neutron capture therapy (BNCT) provides a promising way to destroy cancer cells without damaging healthy tissue. However, BNCT in practice is still limited due to the lack of boron-containing compounds that selectively deliver boron to cancer cells. Since many neuroendocrine tumors show an overexpression of the somatostatin receptor, it was our aim to synthesize compounds that contain a large number of boron atoms and still show high affinity toward this transmembrane receptor. The synthetic peptide Tyr (3)-octreotate (TATE) was chosen as a high-affinity and internalizing tumor targeting vector (TTV). Novel boron cluster compounds, containing 10 or 20 boron atoms, were coupled to the N-terminus of TATE. The obtained affinity data demonstrate that the use of a spacer between TATE and the closo-borane moiety is the option to avoid a loss of biological affinity of closo-borane conjugated TATE. For the first time, it was shown that closo-borane conjugated regulatory peptides retain high biological affinity and selectivity toward their transmembrane tumor receptors. The results obtained and the improvement of spacer and boron building block chemistry may stimulate new directions for BNCT

18F-click labeling and preclinical evaluation of a new 18F-folate for PET imaging

Background The folate receptor (FR) is a well-established target for tumor imaging and therapy. To date, only a few 18 F-folate conjugates via 18 F-prosthetic group labeling for positron emission tomography (PET) imaging have been developed. To some extent, they all lack the optimal balance between efficient radiochemistry and favorable in vivo characteristics. Methods A new clickable olate precursor was synthesized by regioselective coupling of folic acid to 11-azido-3,6,9-trioxaundecan-1-amine at the γ-position of the glutamic acid residue. The non-radioactive reference compound was synthesized via copper-catalyzed azide-alkyne cycloaddition of 3-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)prop-1-yne and γ-(11-azido-3,6,9-trioxaundecanyl)folic acid amide. The radiosynthesis was accomplished in two steps: at first a 18 F-fluorination of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl-4-methylbenzenesulfonate, followed by a 18 F-click reaction with the γ-azido folate. The in vitro, ex vivo, and in vivo behaviors of the new 18 F-folate were investigated using FR-positive human KB cells in displacement assays and microPET studies using KB tumor-bearing mice. Results The new 18 F-folate with oligoethylene spacers showed reduced lipophilicity in respect to the previously developed 18 F-click folate with alkyl spacers and excellent affinity (Ki = 1.6 nM) to the FR. Combining the highly efficient 18 F-click chemistry and a polar oligoethylene-based 18 F-prosthetic group facilitated these results. The overall radiochemical yield of the isolated and formulated product averages 8.7%. In vivo PET imaging in KB tumor-bearing mice showed a tumor uptake of 3.4% ID/g tissue, which could be reduced by FR blockade with native folic acid. Although the new 18 F-oligoethyleneglycole (OEG)-folate showed reduced hepatobiliary excretion over time, a distinct unspecific abdominal background was still observed. Conclusions A new 18 F-folate was developed, being available in very high radiochemical yields via a fast and convenient two-step radiosynthesis. The new 18 F-OEG-folate showed good in vivo behavior and lines up with several recently evaluated 18 F-labeled folates

Injected Human Muscle Precursor Cells Overexpressing PGC-1a Enhance Functional Muscle Regeneration after Trauma

While many groups demonstrated new muscle tissue formation after muscle precursor cell (MPC) injection, the capacity of these cells to heal muscle damage, for example, sphincter in stress urinary incontinence, in long-term is still limited. Therefore, the first goal of our project was to optimize the functional regenerative potential of hMPC by genetic modification to overexpress human peroxisome proliferator-activated receptor gamma coactivator 1-alpha (hPGC-1; α; ), key regulator of exercise-mediated adaptation. Moreover, we aimed at establishing a feasible methodology for noninvasive PET visualization of implanted cells and their microenvironment in muscle crush injury model. PGC-1; α; -bioengineered muscles showed enhanced marker expression for myogenesis (; α; -actinin, MyHC, and Desmin), vascularization (VEGF), neuronal (ACHE), and mitochondrial (COXIV) activity. Consistently, use of hPGC-1; α; _hMPCs produced significantly increased contractile force one to three weeks postinjury. PET imaging showed distinct differences in radiotracer signals ([; 18; F]Fallypride and [; 11; C]Raclopride (both targeting dopamine 2 receptors (D2R)) and [; 64; Cu]NODAGA-RGD (targeting neovascularization)) between GFP_hMPCs and hD2R_hPGC-1; α; _hMPCs. After muscle harvesting, inflammation levels were in parallel to radiotracer uptake amount, with significantly lower uptake in hPGC-1; α; overexpressing samples. In summary, we facilitated early functional muscle tissue regeneration, introducing a novel approach to improve skeletal muscle regeneration. Besides successful tracking of hMPCs in muscle crush injuries, we showed that in high-inflammation areas, the specificity of radioligands might be significantly reduced, addressing a possible bottleneck of neovascularization PET imaging

Evaluation of [11C]Me-NB1 as a potential PET radioligand for measuring GluN2B-containing NMDA receptors, drug occupancy and receptor crosstalk

Clinical and preclinical research with modulators binding to the NMDA receptor GluN2B N-terminal domain (NTD) aim for the treatment of various neurological diseases. However, the interpretation of the results is hampered by the lack of a suitable NMDA PET tracer for assessing the receptor occupancy of candidate drugs. We have developed [11C]Me-NB1 as a PET tracer for imaging GluN1/GluN2B-containing NMDA receptors and used it to investigate in rats the dose-dependent receptor occupancy of eliprodil, a GluN2B NTD modulator. Methods: [11C]Me-NB1 was synthesized and characterized by in vitro displacement binding experiments with rat brain membranes, in vitro autoradiography, blocking and displacement experiments by PET and PET kinetic modeling with an arterial input function. Receptor occupancy by eliprodil was studied in vivo by PET with [11C]Me-NB1. Results: [11C]Me-NB1 was synthesized at 290±90 GBq/µmol specific activity, 7.4±1.9 GBq total activity at the end of synthesis and >99% radiochemical purity. [11C]Me-NB1 binding in rat brain was blocked in vitro and in vivo by the NTD modulators Ro-25-6981 and eliprodil. In vivo, half maximal receptor occupancy by eliprodil occurred at 1.5 μg/kg. At 1 mg/kg eliprodil, a dose with reported neuroprotective effects, >99.5% binding sites were occupied. In vitro, [11C]Me‑NB1 binding was independent of sigma 1 receptor (Sigma1R) and the Sigma1R agonist (+)‑pentazocine did not compete for high affinity binding. In vivo, 2.5 mg/kg (+)‑pentazocine abolished [11C]Me-NB1 specific binding, indicating an indirect effect of Sigma1R on [11C]Me-NB1 binding. Conclusion: [11C]Me-NB1 is suitable for the in vivo imaging of NMDA GluN1/GluN2B receptors and the assessment of the receptor occupancy by NTD modulators. GluN1/GluN2B NMDA receptors are fully occupied at neuroprotective doses of eliprodil. Furthermore, [11C]Me-NB1 enables imaging of GluN1/GluN2B NMDA receptor crosstalk