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We deployed an online pain sensitivity questionnaire (PSQ) and an at-home version of the cold pressor test (CPT) in a large genotyped cohort. We performed genome-wide association studies on the PSQ score (25,321 participants) and CPT duration (6853). We identified one new genome-wide significant locus associated with the PSQ score, which was located in the TSSC1 (also known as EIPR1 ) gene (rs58194899, OR = 0.950 [0.933-0.967], P -value = 1.9 × 10 -8 ). Although high pain sensitivity measured by both PSQ and CPT was associated with individual history of chronic and acute pains, genetic correlation analyses surprisingly suggested an opposite direction: PSQ score was inversely genetically correlated with neck and shoulder pain ( rg = -0.71), rheumatoid arthritis (-0.68), and osteoarthritis (-0.38), and with known risk factors, such as the length of working week (-0.65), smoking (-0.36), or extreme BMI (-0.23). Gene-based analysis followed by pathway analysis showed that genome-wide association studies results were enriched for genes expressed in the brain and involved in neuronal development and glutamatergic synapse signaling pathways. Finally, we confirmed that females with red hair were more sensitive to pain and found that genetic variation in the MC1R gene was associated with an increase in self-perceived pain sensitivity as assessed by the PSQ.

Alternative splicing is a potent modifier of protein function. Stromal interaction molecule 1 (Stim1) is the essential activator of store-operated Ca

To determine the feasibility of complex home-based phenotyping, 1,876 research participants from the customer base of 23andMe participated in an online version of a Pain Sensitivity Questionnaire (PSQ) as well as a cold pressor test (CPT) which is used in clinical assessments of pain. Overall our online version of the PSQ performed similarly to the original pen-and-paper version. Construct validity of the PSQ total was demonstrated by internal consistency and consistent discrimination between more and less painful items. Criterion validity was demonstrated by correlation with pain sensitivity as measured by the cold pressor test. Within the same cohort we performed a cold pressor test using a layperson description and household equipment. Comparison with published reports from controlled studies revealed similar distributions of cold pain tolerance times (i.e., time elapsed before removing the hand from the water). Of those who elected to participate in the CPT, a large majority of participants did not report issues with the test procedure or noncompliance to the instructions (97%). We confirmed a large sex difference in CPT thresholds in line with published data, such that women removed their hands from the water at a median of 54.2 seconds, with men lasting for a median time of 82.7 seconds (Kruskal-Wallis statistic, p < 0.0001), but other factors like age or current pain treatment were at most weakly associated, and inconsistently between men and women. We introduce a new paradigm for performing pain testing, called testing@home, that, in the case of cold nociception, showed comparable results to studies conducted under controlled conditions and supervision of a health care professional.SummaryResearch paradigms employing home-based phenotyping are feasible, with both questionnaires and self-administration of a well-established experimental human pain model yielding similar results compared to controlled settings.

Altered pain sensitivity is believed to play an important role in the development of chronic pain, a common debilitating condition affecting an estimated 1 in 5 adults. Pain sensitivity varies broadly between individuals, and it is notoriously difficult to measure at scale in large populations. Although pain sensitivity is known to be moderately heritable, only a small number of genetic studies have been published and they have had limited success in identifying the genetic architecture of pain sensitivity. In this study, we deployed an online pain sensitivity questionnaire (PSQ) and an at-home version of the cold pressor test (CPT) in a large genotyped cohort. We performed genome-wide association studies (GWAS) on the PSQ score (n = 25,321) and CPT duration (n = 6,853). Despite a reasonably large sample size, we identified only one genome-wide significant locus associated with the PSQ score, which was located in the TSSC1 gene (rs58194899, OR = 0.950 [0.933-0.967], P-value = 1.9*10−8). This gene is responsible for intracellular cell trafficking and it could modulate pain sensitivity via changes in neuroplasticity. PSQ score is genetically correlated with chronic and acute pain, including chronic neck and shoulder pain (rg = 0.71), fracture (0.71), rheumatoid arthritis (0.68), osteoarthritis (0.38), and pneumothorax (0.82). PSQ score was also genetically correlated with known risk factors, such as the length of working week (0.65), noisy workplace (0.41), smoking (0.36), or extreme BMI (0.23). Gene-based analysis followed by pathway analysis showed that GWAS results were enriched for genes expressed in the brain, predominantly in the frontal cortex and basal ganglia, and enriched for genes involved in neuronal development and glutamatergic synapse signaling pathways. Finally, we confirmed that females with red hair were more sensitive to pain and found that genetic variation in the MC1R gene was associated with an increase in pain sensitivity as assessed by the PSQ. Overall, we detailed the genetic background of pain sensitivity using scalable at-home procedures that may serve as a blueprint for larger genetic studies.Author SummaryDespite decades of research and many advances in our understanding of the physical, emotional, and psychological aspects, the genetic contribution to pain sensitivity is still largely unknown. We administered a pain sensitivity questionnaire in parallel with an experimental cold pressor test in a large, genotyped cohort of 31,000 research participants, and found a novel genome-wide genetic association in TSSC1. This gene is responsible for intracellular cell trafficking and it could modulate pain sensitivity via changes in neuroplasticity. Overall, the pain sensitivity measurements revealed strong positive genetic correlations with chronic and acute pain conditions, as well as with unhealthy traits and behaviors, such as obesity, smoking, or difficult working conditions. The genetic association results also suggested, perhaps unsurprisingly, that only the brain tissues could be relevant for pain sensitivity, and identified enrichments for glutamatergic synapse pathways. Despite the complexity of pain sensitivity, our study demonstrated that it is now possible to deploy sophisticated pain questionnaires online and perform experimental medicine tests in at-home settings to further study the genetic architecture of pain sensitivity.

Positron emission tomography (PET) radioligands targeting the human translocator membrane protein (TSPO) are broadly used for the investigations of neuroinflammatory conditions associated with neurological disorders. Structural information on the mammalian protein homodimers&mdash, the suggested functional state of the protein&mdash, is limited to a solid-state nuclear magnetic resonance (NMR) study and to a model based on the previously-deposited solution NMR structure of the monomeric mouse protein. Computational studies performed here suggest that the NMR-solved structure in the presence of detergents is not prone to dimer formation and is furthermore unstable in its native membrane environment. We, therefore, propose a new model of the functionally-relevant dimeric form of the mouse protein, based on a prokaryotic homologue. The model, fully consistent with solid-state NMR data, is very different from the previous predictions. Hence, it provides, for the first time, structural insights into this pharmaceutically-important target which are fully consistent with experimental data.

P2X receptors constitute a seven-member family (P2X1-7) of extracellular ATP-gated cation channels of widespread expression. Because P2X receptors have been implicated in neurological, inflammatory and cardiovascular diseases, they constitute promising drug targets. Since the first P2X cDNA sequences became available in 1994, numerous site-directed mutagenesis studies have been conducted to disclose key sites of P2X receptor function and oligomerization. The publication of the 3-A crystal structures of the zebrafish P2X4 (zfP2X4) receptor in the homotrimeric apo-closed and ATP-bound open states in 2009 and 2012, respectively, has ushered a new era by allowing for the interpretation of the wealth of molecular data in terms of specific three-dimensional models and by paving the way for designing more-decisive experiments. Thanks to these structures, the last five years have provided invaluable insight into our understanding of the structure and function of the P2X receptor class of ligandgated ion channels. In this review, we provide an overview of mutagenesis studies of the pre- and post-crystal structure eras that identified amino acid residues of key importance for ligand binding, channel gating, ion flow, formation of the pore and the channel gate, and desensitization. In addition, the sites that are involved in the trimerization of P2X receptors are reviewed based on mutagenesis studies and interface contacts that were predicted by the zfP2X4 crystal structures.

The P2X7 receptor (P2X7R) belongs to the P2X family of ATP-gated cation channels. P2X7Rs are expressed in epithelial cells, leukocytes, and microglia, and they play important roles in immunological and inflammatory processes. P2X7Rs are obligate homotrimers, with each subunit having two transmembrane helices, TM1 and TM2. Structural and functional data regarding the P2X2 and P2X4 receptors indicate that the central trihelical TM2 bundle forms the intrinsic transmembrane channel of P2X receptors. Here, we studied the accessibility of single cysteines substituted along the pre-TM2 and TM2 helix (residues 327-357) of the P2X7R using as readouts (i) the covalent maleimide fluorescence accessibility of the surface-bound P2X7R and (ii) covalent modulation of macroscopic and single-channel currents using extracellularly and intracellularly applied methanethiosulfonate (MTS) reagents. We found that the channel opening extends from the pre-TM2 region through the outer half of the trihelical TM2 channel. Covalently adducted MTS ethylammonium+ (MTSEA+) strongly increased the probability that the channel was open by delaying channel closing of seven of eight responsive human P2X7R (hP2X7R) mutants. Structural modeling, as supported by experimental probing, suggested that resulting intraluminal hydrogen bonding interactions stabilize the open-channel state. The additional decrease in single-channel conductance by MTSEA+ in five of seven positions identified Y336, S339, L341C, Y343, and G345 as the narrowest part of the channel lumen. The gate and ion-selectivity filter of the P2X7R could be colocalized at and around residue S342. None of our results provided any evidence for dilation of the hP2X7R channel on sustained stimulation with ATP4.

In a previous communication, our efforts leading from 1 to the identification of spiro[cyclohexane-dihydropyrano[3,4-b]indole]-amine 2a as analgesic NOP and opioid receptor agonist were disclosed and their favorable in vitro and in vivo pharmacological properties revealed. We herein report our efforts to further optimize lead 2a, toward trans-6'-fluoro-4',9'-dihydro-N,N-dimethyl-4-phenyl-spiro[cyclohexane-1,1'(3'H)-pyrano[3,4-b]indol]-4-amine (cebranopadol, 3a), which is currently in clinical development for the treatment of severe chronic nociceptive and neuropathic pain.

We report the discovery of spiro[cyclohexane-pyrano[3,4-b]indole]-amines, as functional nociceptin/orphanin FQ peptide (NOP) and opioid receptor agonists with strong efficacy in preclinical models of acute and neuropathic pain. Utilizing 4-(dimethylamino)-4-phenylcyclo-hexanone 1 and tryptophol in an oxa-Pictet-Spengler reaction led to the formation of spiroether 2, representing a novel NOP and opioid peptide receptor agonistic chemotype. This finding initially stems from the systematic derivatization of 1, which resulted in alcohols 3-5, ethers 6 and 7, amines 8-10, 22-24, and 26-28, amides 11 and 25, and urea 12, many with low nanomolar binding affinities at the NOP and mu opioid peptide (MOP) receptors.

Store-operated Ca2+ entry mediated by STIM1-gated Orai1 channels is essential to activate immune cells and its inhibition or gain-of-function can lead to immune dysfunction and other pathologies. Reactive oxygen species interacting with cysteine residues can alter protein function. Pretreatment of the Ca2+ selective Orai1 with the oxidant H2O2 reduces ICRAC with C195, distant to the pore, being its major redox sensor. However, the mechanism of inhibition remained elusive. Here we combine experimental and theoretical approaches and show that oxidation of Orai1 leads to reduced subunit interaction, slows diffusion and that either oxidized C195 or its oxidomimetic mutation C195D located at the exit of transmembrane helix 3 virtually eliminates channel activation by intramolecular interaction with S239 of transmembrane helix 4, thereby locking the channel in a closed conformation. Our results demonstrate a novel mechanistic model for ROS-mediated inhibition of Orai1 and identify a candidate residue for pharmaceutical intervention.

The human P2X7 receptor (hP2X7R) is an ATP-gated nonspecific cation channel. It is expressed in cells of the immune system. Via this receptor, ATP, released during cell damage or hypoxia, serves as danger associated molecular pattern to induce inflammatory reactions. In comparison to other P2X receptors, P2X7R-dependent ionic currents differ in their activation kinetics by an absence of desensitization and induction of large membrane pores by long lasting agonist applications, i.e. the gating and permeation mechanisms of P2X7 receptors are not fully understood.G338 within the second transmembrane domain (TM2) is part of the narrow ion channel pore domain. Substitution of G338 to cysteine reversed the gating behaviour of the hP2X7R channel, i.e. hP2X7G338C expressing Xenopus oocytes displayed a leak conductance which is decreased by application of ATP or by the P2X7R agonist, BzATP. Substitution of extracellular Na+ by the larger organic Tris+ significantly decreased the ion conductance and shifted the reversal potential of the ATP-induced current to more negative potentials indicating that the leak current was carried by cations.Modeling the structural changes of hP2X7G338C during ATP application with the recently published truncated zebrafish P2X4 receptor model as template we found that G338 is located at the narrowest part of the closed ion channel pore and substitution by cysteine pushes the TM2 of the three subunits apart leading to an incomplete channel closure. ATP application leads to a conformation switch of hP2X7G338C which allows an interaction of C338 with Y40 and Y343 decreasing the ion channel pore diameter and leading to a reduced ion current.These findings point to a critical role of G338 in gating the P2X7 receptor.This work was supported by the German research foundation (DFG, Ma 1581/15-1, Schm 536/9-1

Retro-cycloadditions and thermal rearrangements of hydrocarbons have been investigated with density functional theory (B3LYP) and complete active space multiconfiguration self consistent field theory (CASSCF and CASPT2). We review recent results from our laboratories. Subjects are the laser-induced retro-Diels- Alder reactions of norbornene, thermal retro-Diels-Alder reactions of norbornenes and isopropylidenorbornene, 1,3-sigmatropic shifts of vinylcyclopropane and bicyclo(3.2.0)heptenes, and the 1,5-sigmatropic shifts of norcaradienes. The competition between concerted and stepwise processes and the nature of diradicals formed in these processes are described.

A comparative study on the reaction of (CO)3Mo(CH3CN)3 with a series of homologous chiral and prochiral trisphosphines is presented. Only one of the tripodal ligands formed the desired unique complex (CO)3Mo(η3-triphos) (3b) being the first example of a Mo(0)-complex containing a seven- as well as an eight-membered chelate ring. Spectroscopic properties of this complex and two different η2-coordinated species are discussed. The molecular structure of the η3-coordinated complex 3b has been determined by X-ray crystallography.

Transcripts and/or proteins of P2X receptor (P2XR) subunits have been found in virtually all mammalian tissues. Generally more than one of the seven known P2X subunits have been identified in a given cell type. Six of the seven cloned P2X subunits can efficiently form functional homotrimeric ion channels in recombinant expression systems. This is in contrast to other ligand-gated ion channel families, such as the Cys-loop or glutamate receptors, where homomeric assemblies seem to represent the exception rather than the rule. P2XR mediated responses recorded from native tissues rarely match exactly the biophysical and pharmacological properties of heterologously expressed homomeric P2XRs. Heterotrimerization of P2X subunits is likely to account for this observed diversity. While the existence of heterotrimeric P2X2/3Rs and their role in physiological processes is well established, the composition of most other P2XR heteromers and/or the interplay between distinct trimeric receptor complexes in native tissues is not clear. After a description of P2XR assembly and the structure of the intersubunit ATP-binding site, this review summarizes the distribution of P2XR subunits in selected mammalian cell types and the biochemically and/or functionally characterized heteromeric P2XRs that have been observed upon heterologous co-expression of P2XR subunits. We further provide examples where the postulated heteromeric P2XRs have been suggested to occur in native tissues and an overview of the currently available pharmacological tools that have been used to discriminate between homo- and heteromeric P2XRs.

The complexation behaviour of cis- und trans-3-diphenylphosphino-4-hydroxyl-tetrahydrofurans with [Rh(COD)2]BF4 is studied with the help of NMR and IR spectroscopy. In dependence of the spatial arrangement of hydroxyl and phosphino group the formation of different intra- and intermolecular bridged O-P complexes has been observed.

In this paper, we study the classifications of unbalanced data sets of drugs. As an example we chose a data set of 2D6 inhibitors of cytochrome P450. The human cytochrome P450 2D6 isoform plays a key role in the metabolism of many drugs in the preclinical drug discovery process. We have collected a data set from annotated public data and calculated physicochemical properties with chemoinformatics methods. On top of this data, we have built classifiers based on machine learning methods. Data sets with different class distributions lead to the effect that conventional machine learning methods are biased toward the larger class. To overcome this problem and to obtain sensitive but also accurate classifiers we combine machine learning and feature selection methods with techniques addressing the problem of unbalanced classification, such as oversampling and threshold moving. We have used our own implementation of a support vector machine algorithm as well as the maximum entropy method. Our feature selection is based on the unsupervised McCabe method. The classification results from our test set are compared structurally with compounds from the training set. We show that the applied algorithms enable the effective high throughput in silico classification of potential drug candidates.