Strukturbasierte Entwicklung allosterischer Inhibitoren der Myosin Motorfunktion

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Lipase-catalyzed chemoselective ester hydrolysis of biomimetically coupled aryls for the synthesis of unsymmetric biphenyl esters

Lipases are among the most frequently used biocatalysts in organic synthesis, allowing numerous environmentally friendly and inexpensive chemical transformations. Here, we present a biomimetic strategy based on iron(III)-catalyzed oxidative coupling and selective ester monohydrolysis using lipases for the synthesis of unsymmetric biphenyl-based esters under mild conditions. The diverse class of biphenyl esters is of pharmaceutical and technical relevance. We explored the potency of a series of nine different lipases of bacterial, fungal, and mammalian origin on their catalytic activities to cleave biphenyl esters, and optimized the reaction conditions, in terms of reaction time, temperature, pH, organic solvent, and water-organic solvent ratios, to improve the chemoselectivity, and hence control the ratio of unsymmetric versus symmetric products. Elevated temperature and increased DMSO content led to an almost exclusive monohydrolysis by the four lipases Candida rugosa lipase (CRL), Mucor miehei lipase (MML), Rhizopus niveus lipase (RNL), and Pseudomonas fluorescens lipase (PFL). The study was complemented by in silico binding predictions to rationalize the observed differences in effcacies of the lipases to convert biphenyl esters. The optimized reaction conditions were transferred to the preparative scale with high yields, underlining the potential of the presented biomimetic approach as an alternative strategy to the commonly used transition metal-based strategies for the synthesis of diverse biphenyl esters

Analysis of the dominant mutation N188T of human connexin46 (hCx46) using concatenation and molecular dynamics simulation

Connexins (Cx) are proteins that form cell‐to‐cell gap junction channels. A mutation at position 188 in the second extracellular loop (E2) domain of hCx46 has been linked to an autosomal dominant zonular pulverulent cataract. As it is dominantly inherited, it is possible that the mutant variant affects the co‐expressed wild‐type Cx and/or its interaction with other cellular components. Here, we proposed to use concatenated hCx46wt‐hCx46N188T and hCx46N188T‐hCx46wt to analyze how hCx46N188T affected co‐expressed hCx46wt to achieve a dominant inheritance. Heterodimer hCx46wt‐hCx46N188T formed fewer gap junction plaques compared to homodimer hCx46wt‐hCx46wt, while the hCx46N188T‐hCx46N188T homodimer formed almost no gap junction plaques. Dye uptake experiments showed that hemichannels of concatenated variants were similar to hemichannels of monomers. Molecular dynamics simulations revealed that for docking, the N188 of a protomer was engaged in hydrogen bonds (HBs) with R180, N189, and D191 of the counterpart protomer of the adjacent hemichannel. T188 suppressed the formation of HBs between protomers. Molecular dynamics simulations of an equimolar hCx46wt/hCx46N188T gap junction channel revealed a reduced number of HBs between protomers, suggesting reduction of gap junction channels between lens fibers co‐expressing the variants

Correction: Franz et al. Unraveling a Force-Generating Allosteric Pathway of Actomyosin Communication Associated with ADP and Pi Release. Int. J. Mol. Sci. 2021, 22, 104

The author wishes to make the following correction to this paper [...]

C-mannosylation supports folding and enhances stability of thrombospondin repeats

Previous studies demonstrated importance of C-mannosylation for efficient protein secretion. To study its impact on protein folding and stability, we analyzed both C-mannosylated and non-C-mannosylated thrombospondin type 1 repeats (TSRs) of netrin receptor UNC-5. In absence of C-mannosylation, UNC-5 TSRs could only be obtained at low temperature and a significant proportion displayed incorrect intermolecular disulfide bridging, which was hardly observed when C-mannosylated. Glycosylated TSRs exhibited higher resistance to thermal and reductive denaturation processes and the presence of C-mannoses promoted the oxidative folding of a reduced and denatured TSR in vitro. Molecular dynamics simulations supported the experimental studies and showed that C-mannoses can be involved in intramolecular hydrogen bonding and limit the flexibility of the TSR tryptophan-arginine ladder. We propose that in the endoplasmic reticulum folding process, C-mannoses orient the underlying tryptophan residues and facilitate the formation of the tryptophan arginine ladder, thereby influencing the positioning of cysteines and disulfide bridging

Structure and function of the geldanamycin amide synthase from Streptomyces hygroscopicus

Amide synthases catalyze the formation of macrolactam rings from aniline-containing polyketide-derived seco-acids as found in the important class of ansamycin antibiotics. One of these amide synthases is the geldanamycin amide synthase GdmF, which we recombinantly expressed, purified and studied in detail both functionally as well as structurally. Here we show that purified GdmF catalyzes the amide formation using synthetically derived substrates. The atomic structures of the ligand-free enzyme and in complex with simplified substrates reveal distinct structural features of the substrate binding site and a putative role of the flexible interdomain region for the catalysis reaction

Pharmacological inhibitors of the cystic fibrosis transmembrane conductance regulator exert off-target effects on epithelial cation channels

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes

Modern Clinical Research on LSD

All modern clinical studies using the classic hallucinogen lysergic acid diethylamide (LSD) in healthy subjects or patients in the last 25 years are reviewed herein. There were five recent studies in healthy participants and one in patients. In a controlled setting, LSD acutely induced bliss, audiovisual synesthesia, altered meaning of perceptions, derealization, depersonalization, and mystical experiences. These subjective effects of LSD were mediated by the 5-HT2A receptor. LSD increased feelings of closeness to others, openness, trust, and suggestibility. LSD impaired the recognition of sad and fearful faces, reduced left amygdala reactivity to fearful faces, and enhanced emotional empathy. LSD increased the emotional response to music and the meaning of music. LSD acutely produced deficits in sensorimotor gating, similar to observations in schizophrenia. LSD had weak autonomic stimulant effects and elevated plasma cortisol, prolactin, and oxytocin levels. Resting-state functional magnetic resonance studies showed that LSD acutely reduced the integrity of functional brain networks and increased connectivity between networks that normally are more dissociated. LSD increased functional thalamocortical connectivity and functional connectivity of the primary visual cortex with other brain areas. The latter effect was correlated with subjective hallucinations. LSD acutely induced global increases in brain entropy that were associated with greater trait openness 14 days later. In patients with anxiety associated with life-threatening disease, anxiety was reduced for 2 months after two doses of LSD. In medical settings, no complications of LSD administration were observed. These data should contribute to further investigations of the therapeutic potential of LSD in psychiatry

Effective Connectivity of Thalamocortical Interactions Following d-Amphetamine, LSD, and MDMA Administration

BACKGROUND: While the exploration of serotonergic psychedelics as psychiatric medicines deepens, so does the pressure to better understand how these compounds act on the brain. METHODS: We used a double-blind, placebo-controlled, crossover design and administered lysergic acid diethylamide (LSD), 3,4-methylenedioxymethamphetamine (MDMA), and d-amphetamine in 25 healthy participants. By using spectral dynamic causal modeling, we mapped substance-induced changes in effective connectivity between the thalamus and different cortex types (unimodal vs. transmodal) derived from a previous study with resting-state functional magnetic resonance imaging data. Due to the distinct pharmacological modes of action of the 3 substances, we were able to investigate specific effects mainly driven by different neurotransmitter systems on thalamocortical and corticothalamic interactions. RESULTS: Compared with placebo, all 3 substances increased the effective connectivity from the thalamus to specific unimodal cortices, whereas the influence of these cortices on the thalamus was reduced. These results indicate increased bottom-up and decreased top-down information flow between the thalamus and some unimodal cortices. However, for the amphetamines, we found the opposite effects when examining the effective connectivity with transmodal cortices, including parts of the salience network. Intriguingly, LSD increased the effective connectivity from the thalamus to both unimodal and transmodal cortices, indicating a breach in the hierarchical organization of ongoing brain activity. CONCLUSIONS: The results advance our knowledge about the action of psychedelics on the brain and refine current models aiming to explain the underlying neurobiological processes