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−.
To determine the feasibility of complex home-based phenotyping, 1,876 research participants from the customer base of 23andMe completed 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 CPT. 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 with 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.
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.
Atomistic descriptions of the μ-opioid receptor (μOR) noncovalently binding with two of its prototypical morphinan agonists, morphine (MOP) and hydromorphone (HMP), are investigated using molecular ...dynamics (MD) simulations. Subtle differences between the binding modes and hydration properties of MOP and HMP emerge from the calculations. Alchemical free energy perturbation calculations show qualitative agreement with in vitro experiments performed in this work: indeed, the binding free energy difference between MOP and HMP computed by forward and backward alchemical transformation is 1.2±1.1 and 0.8±0.8 kcal/mol, respectively, to be compared with 0.4±0.3 kcal/mol from experiment. Comparison with an MD simulation of μOR covalently bound with the antagonist β-funaltrexamine hints to agonist-induced conformational changes associated with an early event of the receptor's activation: a shift of the transmembrane helix 6 relative to the transmembrane helix 3 and a consequent loss of the key R165-T279 interhelical hydrogen bond. This finding is consistent with a previous proposal suggesting that the R165-T279 hydrogen bond between these two helices indicates an inactive receptor conformation.
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-the suggested functional state of the protein-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.
In a previous communication, our efforts leading from 1 to the identification of spirocyclohexane-dihydropyrano3,4-bindole-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-spirocyclohexane-1,1′(3′H)-pyrano3,4-bindol-4-amine (cebranopadol, 3a), which is currently in clinical development for the treatment of severe chronic nociceptive and neuropathic pain.
The homotrimeric P2X3 receptor, one of the seven members of the ATP-gated P2X receptor family, plays a crucial role in sensory neurotransmission. P2X3 receptor antagonists have been identified as ...promising drugs to treat chronic cough and are suggested to offer pain relief in chronic pain such as neuropathic pain. Here, we analysed whether compounds affect P2X3 receptor activity by high-throughput screening of the Spectrum Collection of 2000 approved drugs, natural products and bioactive substances. We identified aurintricarboxylic acid (ATA) as a nanomolar-potency antagonist of P2X3 receptor-mediated responses. Two-electrode voltage clamp electrophysiology-based concentration–response analysis and selectivity profiling revealed that ATA strongly inhibits the rP2X1 and rP2X3 receptors (with IC50 values of 8.6 nM and 72.9 nM, respectively) and more weakly inhibits P2X2/3, P2X2, P2X4 or P2X7 receptors (IC50 values of 0.76 μM, 22 μM, 763 μM or 118 μM, respectively). Patch-clamp analysis of mouse DRG neurons revealed that ATA inhibited native P2X3 and P2X2/3 receptors to a similar extent than rat P2X3 and P2X2/3 receptors expressed in Xenopus oocytes. In a radioligand binding assay, up to 30 μM ATA did not compete with 3H-ATP for rP2X3 receptor binding, indicating a non-competitive mechanism of action. Molecular docking studies, site-directed mutagenesis and concentration–response analysis revealed that ATA binds to the negative allosteric site of the hP2X3 receptor. In summary, ATA as a drug-like pharmacological tool compound is a nanomolar-potency, allosteric antagonist with selectivity towards αβ-methylene-ATP-sensitive P2X1 and P2X3 receptors.
•P2X3R activity of 2000 approved drugs, natural products and bioactive substances.•Aurintricarboxylic acid (ATA) is a nanomolar-potency antagonist of P2X1R and P2X3R.•ATA as a allosteric antagonist binds to the negative allosteric site of the hP2X3R.•ATA as a drug-like pharmacological tool compound fulfils Lipinski's rule of five.•ATA blocks P2X3R of DRG neurons and is suitable for in vivo analysis of the P2X3R.
Blockade of voltage‐gated sodium channels (VGSCs) has been used successfully in the clinic to enable control of pathological firing patterns that occur in conditions as diverse as chronic pain, ...epilepsy, and arrhythmias. Herein we review the state of the art in marketed sodium channel inhibitors, including a brief compendium of their binding sites and of the cellular and molecular biology of sodium channels. Despite the preferential action of this drug class toward over‐excited cells, which significantly limits potential undesired side effects on other cells, the need to develop a second generation of sodium channel inhibitors to overcome their critical clinical shortcomings is apparent. Current approaches in drug discovery to deliver novel and truly innovative sodium channel inhibitors is next presented by surveying the most recent medicinal chemistry breakthroughs in the field of small molecules and developments in automated patch‐clamp platforms. Various strategies aimed at identifying small molecules that target either particular isoforms of sodium channels involved in specific diseases or anomalous sodium channel currents, irrespective of the isoform by which they have been generated, are critically discussed and revised.
Nav1 in the crosshairs: Herein we review the current state of the art in voltage‐gated sodium channels as drug targets, including the molecular mechanisms that underlie sodium channel modulators. Recent developments in drug discovery aimed at the identification of a new generation of small‐molecule inhibitors, including the pharmacological exploitation of other binding sites, are also discussed.
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