Structure-Guided Design of Nurr1 Agonists Derived from the Natural Ligand Dihydroxyindole.

The neuroprotective transcription factor Nurr1 was recently found to bind the dopamine metabolite 5,6-dihydroxyindole (DHI) providing access to Nurr1 ligand design from a natural template. We screened a custom set of 14 k extended DHI analogues in silico for optimized descendants to select 24 candidates for microscale synthesis and in vitro testing. Three out of six primary hits were validated as novel Nurr1 agonists with up to sub-micromolar binding affinity, highlighting the druggability of the Nurr1 surface region lining helix 12. In vitro profiling confirmed cellular target engagement of DHI descendants and demonstrated remarkable additive effects of combined Nurr1 agonist treatment, indicating diverse binding sites mediating Nurr1 activation, which may open new avenues in Nurr1 modulation.

Fragment-based discovery of orphan nuclear receptor Nur77/NGFI-B ligands.

The transcription factor nerve growth factor-induced clone B (NGFI-B, Nur77, NR4A1) is an orphan nuclear receptor playing a role in cell survival and apoptosis regulation. Pharmacological Nur77 modulation holds promise for cancer and (neuro-)inflammatory disease treatment. The available Nur77 ligand scaffolds based on highly lipophilic natural products cytosporone B, celastrol and isoalantolactone are inadequate for the development of potent Nur77 modulators with favorable properties as chemical tools and future drugs. By fragment library screening and subsequent modeling for fragment extension, we have obtained a set of new Nur77 ligands offering alternative chemotypes for the development of Nur77 agonists and inverse agonists. Computer-aided fragment extension in a second stage screening yielded a Nur77 agonist with significant activation efficacy and preference over the related NR4A receptors.

Self-reporting styrylthiazolium photopharmaceuticals: mitochondrial localisation as well as SAR drive biological activity.

Novel photoswitches offering features complementary to the well-established azobenzenes are increasingly driving high-precision research in cellular photopharmacology. Styrylthiazolium (StyTz) and styrylbenzothiazolium (StyBtz) are cellularly untested E/Z-isomerisation photoswitches which are nearly isosteric to azobenzenes, but have distinct properties: including ca. 60 nm red-shifted π → π* absorption, self-reporting fluorescence, Z → E relaxation on typical biological timescales, and decent solubility (positive charge). We tested StyTz and StyBtz for their potential as photopharmaceutical scaffolds, by applying them to photocontrol microtubule dynamics. They light-specifically disrupt microtubule network architecture and block cell proliferation: yet, testing lead compound StyBtz2 for its molecular mechanism of action showed that it did not inhibit microtubule dynamics. Using its self-reporting fluorescence, we tracked its localisation in live cells and observed accumulation of E-StyBtz2 into mitochondria; during prolonged illumination, it was released into the cytosol, and blebbing and cell death were observed. We interpret this as light-dependent rupturing of mitochondria on acute timescales. We conclude that StyTz/StyBtz can be interesting photopharmaceutical scaffolds for addressing mitochondrial, rather than cytosolic, targets.

[Is Mandatory Vaccination Against the Coronavirus Necessary?] / Ist eine Impfpflicht gegen das Coronavirus nötig?

The end of the pandemic requires that SARS-CoV-2 vaccines be used. However, vaccination itself can lead to temporary adverse health effects and/or long-term damage. Vaccination initially represents a private good demanded by an individual cost-benefit calculus; however, it also creates positive externalities and thus too few individual incentives to vaccinate. Getting vaccinated is not a dominant rational strategy, neither in the overall population, nor among the old and the young, nor when the long-term costs of the pandemic are taken into account. It is all the more important to "price in" the long-term consequences of a lasting pandemic.

New Resensitizers for the Nicotinic Acetylcholine Receptor by Ligand-Based Pharmacophore Modeling.

INTRODUCTION: Irreversible inhibition of the acetylcholinesterase upon intoxication with organophosphorus compounds leads to an accumulation of acetylcholine in the synaptic cleft and a subsequent desensitization of nicotinic acetylcholine receptors which may ultimately result in respiratory failure. A direct intervention at the nicotinic acetylcholine receptor (nAChR) was proposed as an alternative therapeutic approach to the treatment with atropine and oximes. METHODS: The bispyridinium compound MB327 has been found to recover functional activity of nAChR thus representing a promising starting point for the development of new drugs for the treatment of organophosphate poisoning. Recent solid-supported membrane-based electrophysiological experiments have identified symmetrically substituted bispyridinium compounds e.g. MB327, MB583, and PTM0001 that are able to resensitize nAChR of Torpedo californica. In addition, six compounds have been found not to show any resensitizing potential and were thus classified as inactive. This set of active and inactive bispyridinium compounds was taken to develop a pharmacophore model and in silico screening of a virtual database of bispyridinium compounds to identify new compounds that are able to restore the functional activity of desensitized nAChR. RESULTS: Screening of a virtual compound database of symmetrically substituted bispyridinium compounds with the derived pharmacophore yielded several promising compounds which satisfy the pharmacophore and ought to have the same or even better resensitizing effect on nAChR as the parent compound MB327.

Synthesis of a Series of Non-Symmetric Bispyridinium and Related Compounds and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor.

The current standard therapy to counteract organophosphate intoxication is not effective in equal measure against all types of organophosphorus compounds (OPCs), as the outcome of oxime-induced reactivation of inactivated acetylcholinesterase (AChE) strongly depends on the particular OPC. In case the reactivation is insufficient, acetylcholine concentrations that rise to pathophysiological levels force the nicotinic acetylcholine receptor (nAChR) into a desensitized state and hence a functionally inactive state. As a consequence, neurotransmission is irreversibly disrupted at the neuromuscular junction. Previous electrophysiological studies identified the symmetric bispyridinium compound 1,1'-(propane-1,3-diyl)bis[4-(tert-butyl)pyridin-1-ium] diiodide (MB327) as a re-sensitizer of the desensitized nAChR. MB327 is thereby capable of restoring the functional activity. Very recently, in silico modeling studies suggested non-symmetric derivatives of MB327 as potential re-sensitizers with enhanced binding affinity and thus possible enhanced efficacy. In this study, 26 novel non-symmetric bispyridinium compounds and related derivatives were synthesized. For the synthesis of the highly polar target compounds in sufficient quantities, newly developed and highly efficient two-step procedures were used. Compounds were characterized in terms of their binding affinity toward the MB327 binding site at the nAChR using recently developed mass spectrometry (MS) Binding Assays. Regarding structure-affinity relationships at the MB327 binding site, the presence of two quaternary aromatic nitrogen centers as well as pyridinium systems with a tert-butyl group at the 4-position or a NMe2 group at the 3- or 4-positions appeared to be beneficial for high binding affinities.

Novel Allosteric Ligands of γ-Aminobutyric Acid Transporter 1 (GAT1) by MS Based Screening of Pseudostatic Hydrazone Libraries.

This study describes the screening of dynamic combinatorial libraries based on nipecotic acid as core structure with substituents attached to the 5- instead of the common 1-position for the search of novel inhibitors of the GABA transporter GAT1. The generated pseudostatic hydrazone libraries included a total of nearly 900 compounds and were screened for their binding affinities toward GAT1 in competitive mass spectrometry (MS) based Binding Assays. Characterization of the hydrazones with the highest affinities (with cis-configured rac-16gf bearing a 5-(1-naphthyl)furan-2-yl residue and a four atom spacer being the most potent) in binding and uptake experiments revealed an allosteric interaction at GAT1, which was not reported for any other nipecotic acid derivative up to now. Therefore, the herein introduced 5-substituted nipecotic acid derivatives could serve as valuable tools for investigations of allosterically modulated GABA transport mediated by GAT1 and furthermore as starting point for a new class of GAT1 inhibitors.

Synthesis of a Series of Structurally Diverse MB327 Derivatives and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor.

A novel series of 30 symmetric bispyridinium and related N-heteroaromatic bisquaternary salts with a propane-1,3-diyl linker was synthesized and characterized for their binding affinity at the MB327 binding site of nicotinic acetylcholine receptor (nAChR) from Torpedo californica. Compounds targeting this binding site are of particular interest for research into new antidotes against organophosphate poisoning, as therapeutically active 4-tert-butyl-substituted bispyridinium salt MB327 was previously identified as a nAChR re-sensitizer. Efficient access to the target compounds was provided by newly developed methods enabling N-alkylation of sterically hindered or electronically deactivated heterocycles exhibiting a wide variety of functional groups. Determination of binding affinities toward the MB327 binding site at the nAChR, using a recently developed mass spectrometry (MS)-based Binding Assay, revealed that several compounds reached affinities similar to that of MB327 (pKi =4.73±0.03). Notably, the newly prepared lipophilic 4-tert-butyl-3-phenyl-substituted bispyridinium salt PTM0022 (3 h) was found to have significantly higher binding affinity, with a pKi value of 5.16±0.07, thus representing considerable progress toward the development of more potent nAChR re-sensitizers.

Development of New Photoswitchable Azobenzene Based γ-Aminobutyric Acid (GABA) Uptake Inhibitors with Distinctly Enhanced Potency upon Photoactivation.

A series of nipecotic acid derivatives with new azo benzene based photoswitchable N-substituents was synthesized and characterized in their ( E)- and ( Z)-form for their functional inhibitory activity at γ-aminobutyric acid transporters subtype 1 (GAT1), the most common γ-aminobutyric acid (GABA) transporter subtype in the central nervous system (CNS). This led to the identification of the first photoswitchable ligands exhibiting a moderate uptake inhibition of GABA in their ( E)- but distinctive higher inhibitory potency in their ( Z)-form resulting from photoirradiation. For the most efficient photoactivatable nipecotic acid derivative displaying an N-but-3-yn-1-yl linker with a terminal diphenyldiazene unit, an inhibitory potency of 4.65 ± 0.05 (pIC50) was found for its ( E)-form. which increased by almost two log units up to 6.38 ± 0.04 when irradiated. The effect of this photoswitchable mGAT1 inhibitor has also been evaluated and confirmed in patch-clamp recordings in acute hippocampal slices from mice.

Searching for putative binding sites of the bispyridinium compound MB327 in the nicotinic acetylcholine receptor.

Irreversible inhibition of the acetylcholine esterase upon intoxication with organophosphorus compounds leads to an accumulation of acetylcholine in the synaptic cleft and a subsequent desensitization of nicotinic acetylcholine receptors which may ultimately result in respiratory failure. The bispyridinium compound MB327 has been found to restore functional activity of nAChR thus representing a promising starting point for the development of new drugs for the treatment of organophosphate poisoning. In order to optimize the resensitizing effect of MB327 on nAChR, it would be very helpful to know the MB327 specific binding site to apply structure based molecular modeling. The binding site for MB327 at the nAChR is not known and so far goal of speculations, but it has been shown that MB327 does not bind to the orthosteric acetylcholine binding site. We have used docking calculations to screen the surface of nAChR for possible binding sites of MB327. The results indicate that at least two potential binding sites for MB327 at nAChR are present inside the channel pore. In these binding sites, MB327 intercalates between the γ-α and ß-δ subunits of nAChR, respectively. Both putative MB327 binding sites show an unsymmetrical distribution of surrounding hydrophilic and lipophilic amino acids. This suggests that substitution of MB327-related bispyridinium compounds on one of the two pyridinium rings with polar substituents should have a favorable effect on the pharmacological function.

Biceps Femoris Injury a Rarity: A Case Report.

Isolated biceps femoris rupture is a rare injury associated with limitation in the function of the knee. We present a 65-year-old man who sustained an isolated complete rupture of the tendon of the biceps femoris. The diagnostic was reached after clinical examination and magnetic resonance imaging of the affected knee. This case was treated with a surgical tendon reconstruction. The outcome was good and the patient was able to walk normally again without limitation, even if he did not comply with our recommendation.

Overcoming resistance against managed care - insights from a bargaining model.

Recent healthcare reforms have sought to increase efficiency by introducing managed care (MC) while respecting consumer preferences by admitting choice between MC and conventional care. This article proposes an institutional change designed to let German consumers choose between the two settings through directing payments from the Federal Health Fund to social health insurers (SHIs) or to specialized MC organizations (MCOs). To gauge the chance of success of this reform, a game involving a SHI, a MCO, and a representative insured (RI) is analyzed. In a "three-player/three-cake" game the coalitions {SHI, MCO}, {MCO, RI}, and {SHI, RI} can form. Players' possibility to switch between coalitions creates new outside options, causing the conventional bilateral Nash bargaining solution to be replaced by the so-called von Neumann-Morgenstern triple. These triples are compared to the status quo (where the RI has no threat potential) and related to institutional conditions characterizing Germany, the Netherlands, and Switzerland.

Development of Highly Potent GAT1 Inhibitors: Synthesis of Nipecotic Acid Derivatives with N-Arylalkynyl Substituents.

A new scaffold of highly potent and mGAT1-selective inhibitors has been developed. Compounds in this class are characterized by an alkyne-type spacer connecting nipecotic acid with an aromatic moiety. Preliminary evaluations made it apparent that a nipecotic acid derivative with an N-butynyl linker and a terminal 2-biphenyl residue exhibiting a binding affinity (pKi ) of 7.61±0.03 to mGAT1 and uptake inhibition (pIC50 ) of 7.00±0.06 selective for mGAT1 could serve as a hit compound. Docking calculations for compounds based on this structure in an hGAT1 homology modeling study indicated binding affinities similar to or even higher than that of the well-known mGAT1 inhibitor tiagabine. Synthesis of the designed compounds was readily carried out by two consecutive cross-coupling reactions, giving flexible access to variously substituted biphenyl subunits. With an appropriate substitution pattern of the biphenyl moiety, the binding affinity of enantiopure (R)-nipecotic acid derivatives to mGAT1 increased to pKi =8.33±0.01, and the uptake inhibitory potency up to pIC50 =7.72±0.02.

Synthesis of 4-substituted nipecotic acid derivatives and their evaluation as potential GABA uptake inhibitors.

In this study, we disclose the design and synthesis of novel 4-susbtituted nipecotic acid derivatives as inhibitors of the GABA transporter mGAT1. Based on molecular modeling studies the compounds are assumed to adopt a binding pose similar to that of the potent mGAT1 inhibitor nipecotic acid. As substitution in 4-position should not cause an energetically unfavorable orientation of nipecotic acid as it is the case for N-substituted derivatives this is expected to lead to highly potent binders. For the synthesis of novel 4-substituted nipecotic acid derivatives a linear synthetic strategy was employed. As a key step, palladium catalyzed cross coupling reactions were used to attach the required biaryl moieties to the ω-position of the alkenyl- or alkynyl spacers of varying length in the 4-position of the nipecotic acid scaffold. The resulting amino acids were characterized with respect to their binding affinities and inhibitory potencies at mGAT1. Though the biological activities found were generally insignificant to poor, two compounds, one of which possesses a reasonable binding affinity for mGAT1, rac-57, the other a notable inhibitory potency at mGAT4, rac-84, both displaying a slight subtype selectivity for the individual transporters, could be identified.

Different Binding Modes of Small and Large Binders of GAT1.

Well-known inhibitors of the γ-aminobutyric acid (GABA) transporter GAT1 share a common scaffold of a small cyclic amino acid linked by an alkyl chain to a moiety with two aromatic rings. Tiagabine, the only FDA-approved GAT1 inhibitor, is a typical example. Some small amino acids such as (R)-nipecotic acid are medium-to-strong binders of GAT1, but similar compounds, such as proline, are very weak binders. When substituted with 4,4-diphenylbut-3-en-1-yl (DPB) or 4,4-bis(3-methylthiophen-2-yl)but-3-en-1-yl (BTB) groups, the resulting compounds have similar pKi and pIC50 values, even though the pure amino acids have very different values. To investigate if small amino acids and their substituted counterparts share a similar binding mode, we synthesized butyl-, DPB-, and BTB-substituted derivatives of small amino acids. Supported by the results of docking studies, we propose different binding modes not only for unsubstituted und substituted, but also for strong- and weak-binding amino acids. These data lead to the conclusion that following a fragment-based approach, not pure but N-butyl-substituted amino acids should be used as starting points, giving a better estimate of the activity when a BTB or DPB substituent is added.

Development of Highly Potent GAT1 Inhibitors: Synthesis of Nipecotic Acid Derivatives by Suzuki-Miyaura Cross-Coupling Reactions.

A new series of potent and selective mGAT1 inhibitors has been identified, featuring a nipecotic acid residue and an N-butenyl linker with a 2-biphenyl residue at the ω-position. Docking, combined with MD calculations, revealed a binding mode for the new compounds similar to that of tiagabine, the only mGAT1 inhibitor currently approved as antiepileptic drug. For the synthesis, a Suzuki-Miyaura cross-coupling reaction was used as a key step by which variously substituted biaryl subunits were assembled. Biological evaluation revealed several compounds that possess binding affinities and inhibitory potencies toward mGAT1, together with subtype selectivities against mGAT2-mGAT4 that were similar to or even higher than those for tiagabine. A derivative carrying the 2',4'-dichloro-2-biphenyl moiety attached to N-but-3-enylnipecotic acid at the terminal position of the linker chain was found to be the most potent binder, with the racemic form of the compound displaying a binding affinity of 8.05±0.13 (pKi ), while the R enantiomer exhibited an affinity value of 8.33±0.06 (pKi ).

The glutamic acid-rich protein is a gating inhibitor of cyclic nucleotide-gated channels.

The cyclic nucleotide-gated (CNG) cation channel of rod photoreceptors is a heterotetramer consisting of homologous CNGA1 and CNGB1a subunits. While CNGA1 is indispensable for channel activation, the specific role of CNGB1a in this process has remained elusive. Here, we show that the N-terminal glutamic acid-rich protein (GARP) domain of CNGB1a and soluble GARP2, which corresponds to the proximal portion of the GARP domain, act as autoinhibitory domains that decrease the opening probability of the CNG channel. In the presence of mutations that structurally impair the cyclic nucleotide-binding domain (CNBD) of CNGB1a, the GARP domain completely abolishes channel activity. In agreement with an inhibitory function of GARP, the activity of mutant CNG channels could be fully restored by deletion of the GARP domain. We identified two sequences within the GARP domain that confer most of the inhibitory effect and demonstrate that the profound inhibition imposed by the GARP domain is caused by direct and autonomous protein-protein interaction with the CNG channel complex. In wild-type rod CNG channels, this inhibitory effect can be relieved by binding of cGMP to the CNBD of CNGB1a. In conclusion, we propose that the N terminus of CNGB1a and soluble GARPs act as molecular gate keepers that control the activation of heteromeric rod CNG channels. Our results suggest that the GARP domain has evolved in rod photoreceptors to reduce current noise resulting from openings of CNG channels in the absence of cGMP.

Generation of a 3D model for human GABA transporter hGAT-1 using molecular modeling and investigation of the binding of GABA.

A three-dimensional model of the human Na(+)/Cl(-)-dependent gamma-aminobutyric acid (GABA) transporter hGAT-1 was developed by homology modeling and refined by subsequent molecular modeling using the crystal structure of a bacterial homologue leucine transporter from Aquifex aeolicus (LeuT(Aa)) as the template. Protein structure quality checks show that the resulting structure is particularly suited for the analysis of the substrate binding pocket and virtual screening experiments. Interactions of GABA and the substrate binding pocket were investigated using docking studies. The difference of 6 out of 13 substrate interacting side chains between hGAT-1 and LeuT(Aa) lead to the different substrate preference which can be explained using our three-dimensional model of hGAT-1. In particular the replacement of serine 256 and isoleucine 359 in LeuT(Aa) with glycine and threonine in hGAT-1 seems to facilitate the selection of GABA as the main substrate by changing the hydrogen bonding pattern in the active site to the amino group of the substrate. For a set of 12 compounds flexible docking experiments were performed using LigandFit in combination with the Jain scoring function. With few exceptions the obtained rank order of potency was in line with experimental data. Thus, the method can be assumed to give at least a rough estimate of the potency of the potential of GABA uptake inhibitors.