In a previous communication, our efforts leading from 1 to the identification of spiro [cyclohexanedihydropyrano[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′-, which is currently in clinical development for the treatment of severe chronic nociceptive and neuropathic pain. KEYWORDS: NOP receptor agonists, MOP receptor agonists, cebranopadol, analgesics R ecent publications indicate that small molecules activating both nociceptin/orphanin FQ peptide (NOP) and mu opioid peptide (MOP) receptors may potentiate opiate analgesia and at the same time display an improved side effects profile. 1,2 We have recently reported the discovery of a series of small molecules, characterized by their high NOP and opioid receptor agonistic activity. 3 This series included uncyclized (e.g., 1) as well as spirocyclic examples (e.g., 2a). The discovery of spirocyclic 2a originated from the respective uncyclized analogues, which were potent NOP and MOP receptor binders but sometimes hampered by only partial agonistic NOP and MOP receptor activity. In particular, the spiroindole derivates sparked our interest due to their structural novelty and favorable in vitro and in vivo properties. The leading spiroether 2a exhibited strong efficacy in preclinical models of acute (ED 50 rat tail-flick: 3.63 nmol/kg i.v.) and neuropathic pain (ED 50 rat spinal nerve ligation: 1.05 nmol/kg i.v.) but was hampered by poor pharmacokinetic (PK) properties in rats with high clearance, large volume of distribution, moderate half-life (Cl = 4.0 L/h·kg; V ss = 7.52 L/kg; t 1/2 = 1.6 h), and a critically very low oral bioavailability (F = 4%).We herein report our efforts to further optimize the spiroindole lead 2a, which eventually led to the discovery of trans-6′-fluoro-4′,9′-dihydro-N,N-dimethyl-4-phenyl-spiro-[cyclohexane-1,1′(3′H)-pyrano [3,4-b]indol]-4-amine (3a, cebranopadol), a novel potent analgesic NOP and opioid receptor agonist, currently in clinical development for the treatment of severe chronic nociceptive and neuropathic pain.The structure−activity relationship (SAR) established around the uncyclized scaffolds (e.g., 1) suggested that a broad variety of linkers such as alcohols, ethers, and amines are tolerated, showing high NOP and MOP receptor binding affinities. 3 As a result, we applied this knowledge through analogous structural variations to lead structure 2a (region A, Figure 1). These changes were also combined with a targeted approach to improve the poor PK profile, in particular by addressing metabolically liable regions B and C.With this in mind, the transformation of the oxacyclic spiro moiety into a carba-, aza-, or thio-cyclic moiety was investigated. Advancing from the oxacyclic spiro 2a to the azacyclic moiety 5a led to equally potent NOP and MOP receptor binders, as well as the introduction of the N-methyl subunit 6a. Similarly, pot...

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. ytolytic pore formation by extracellular adenosine triphosphate (ATP) was first described in mast cells and was outright suggested to involve the activation and subsequent pore dilatation of a hypothetical ATP receptor channel (1). A similar ATP-triggered pore-forming activity was subsequently found in many immune and inflammatory cells and certain transformed cell lines. The distinctive feature that is unique to this tentatively termed P2Z receptor (2) is that brief application of ATP (in its tetraanionic form, ATP 4− ) evokes depolarizing cation fluxes, whereas prolonged application of ATP 4− causes formation of cytolytic pores with a molecular cutoff of ∼900 Da (for review, see ref.3). The cytolytic activity was eventually assigned to the P2X7 receptor (P2X7R), the seventh and final member of the P2X receptor family. In HEK293 cells, the recombinant P2X7R conferred the same responses that were attributed previously to the P2Z receptor, including the dual-mode operation as a cation channel and a cytolytic pore (4, 5). Today, it is widely believed that P2X7R can mediate apoptotic or necrotic cell death under pathophysiological conditions (4-6).The cytolytic activity of human P2X7R (hP2X7R) has been attributed to a time-dependent dilation of the integral ion channel based on macroscopic current recordings of various cell types (7-11). Particularly revealing was the observation that substituting T348 and D352 with basic residues in the channellining second transmembrane domain (TM2) of the rat P2X7R (rP2X7R) simultaneously increased the permeability of the normally cationic channel for Cl − and an acidic fluorescent dye with an effective diameter of >10 Å (12). A "channel-to-pore" dilatation that changed the permeation characteristic from small inorganic cations (e.g., Na + , K + , and Ca 2+) to large organic cations [e.g., N-methyl-D-glucamine (NMDG + )] has also been found for P2X2, P2X2/X3, and P2X4 receptors (13, 14) and certain transient receptor potential (TRP) channels (15, 16). In contrast, in extended single-chann...

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.

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.

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.