Daniel Ursu

Pungency of TRPV1 agonists is directly correlated with kinetics of receptor activation and lipophilicity

TRPV1 (transient receptor potential vanilloid 1) is a ligand-gated ion channel expressed predominantly in nociceptive primary afferents that plays a key role in pain processing. In vivo activation of TRPV1 receptors by natural agonists like capsaicin is associated with a sharp and burning pain, frequently described as pungency. To elucidate the mechanisms underlying pungency we investigated a series of TRPV1 agonists that included both pungent and non-pungent compounds covering a large range of potencies. Pungency of capsaicin, piperine, arvanil, olvanil, RTX (resiniferatoxin) and SDZ-249665 was evaluated in vivo, by determining the increase in the number of eye wipes caused by direct instillation of agonist solutions into the eye. Agonist-induced calcium fluxes were recorded using the FLIPR technique in a recombinant, TRPV1-expressing cell line. Current-clamp recordings were performed in rat DRG (dorsal root ganglia) neurons in order to assess the consequences of TRPV1 activation on neuronal excitability. Using the eye wipe assay the following rank of pungency was obtained: capsaicin>piperine>RTX>arvanil>olvanil>SDZ-249665. We found a strong correlation between kinetics of calcium flux, pungency and lipophilicity of TRPV1 agonists. Current-clamp recordings confirmed that the rate of receptor activation translates in the ability of agonists to generate action potentials in sensory neurons. We have demonstrated that the lipophilicity of the compounds is directly related to the kinetics of TRPV1 activation and that the latter influences their ability to trigger action potentials in sensory neurons and, ultimately, pungency.

Gain and loss of function of P2X7 receptors: mechanisms, pharmacology and relevance to diabetic neuropathic pain

BackgroundGenetic causes of exaggerated or reduced pain sensitivity in humans are well known. Recently, single nucleotide polymorphisms (SNPs) in the gene P2RX7, coding for the ATP-gated ion channel P2X7, have been described that cause gain-of-function (GOF) and loss-of-function (LOF), respectively of this channel. Importantly, P2RX7 SNPs have been associated with more or less severe pain scores in patient suffering of post-mastectomy pain and osteoarthritis.ResultsThe functional consequences of some P2RX7 SNPs (rs208294 (His155Tyr), rs1718119 (Ala348Thr) and rs3751143 (Glu496Ala)) were studied in recombinant cells in vitro. Our findings suggest a correlation between GOF and LOF of P2X7 and actual channel protein expression. Both channel and pore function for these mutant P2X7 receptors changed in parallel to protein levels. On the other hand, the mutant receptors did not differ in their sensitivity to known P2X7 agonists and antagonists. We further demonstrated that in patients with diabetic peripheral neuropathic pain (DPNP), the presence of the GOF SNPs rs208294 (His155Tyr) and rs1718119 (Ala348Thr) is associated, in females, with higher pain intensity scores.ConclusionsOur present results confirm the physiological relevance of some of the SNPs in the P2RX7 gene and show that the presence of these genetic variants correlates with pain sensitivity also in a diabetic neuropathic pain patient population.

Contrasting properties of α7-selective orthosteric and allosteric agonists examined on native nicotinic acetylcholine receptors.

Subtype-selective ligands are important tools for the pharmacological characterisation of neurotransmitter receptors. This is particularly the case for nicotinic acetylcholine receptors (nAChRs), given the heterogeneity of their subunit composition. In addition to agonists and antagonists that interact with the extracellular orthosteric nAChR binding site, a series of nAChR allosteric modulators have been identified that interact with a distinct transmembrane site. Here we report studies conducted with three pharmacologically distinct nicotinic ligands, an orthosteric agonist (compound B), a positive allosteric modulator (TQS) and an allosteric agonist (4BP-TQS). The primary focus of the work described in this study is to examine the suitability of these compounds for the characterisation of native neuronal receptors (both rat and human). However, initial experiments were conducted on recombinant nAChRs demonstrating the selectivity of these three compounds for α7 nAChRs. In patch-clamp recordings on rat primary hippocampal neurons we found that all these compounds displayed pharmacological properties that mimicked closely those observed on recombinant α7 nAChRs. However, it was not possible to detect functional responses with compound B, an orthosteric agonist, using a fluorescent intracellular calcium assay on either rat hippocampal neurons or with human induced pluripotent stem cell-derived neurons (iCell neurons). This is, presumably, due to the rapid desensitisation of α7 nAChR that is induced by orthosteric agonists. In contrast, clear agonist-evoked responses were observed in fluorescence-based assays with the non-desensitising allosteric agonist 4BP-TQS and also when compound B was co-applied with the non-desensitising positive allosteric modulator TQS. In summary, we have demonstrated the suitability of subtype-selective orthosteric and allosteric ligands for the pharmacological identification and characterisation of native nAChRs and the usefulness of ligands that minimise receptor desensitisation for the characterisation of α7 nAChRs in fluorescence-based assays.

α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties.

The existence of α7β2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer’s disease, it is critical to determine whether α7β2 nAChRs are present in the human brain, in which brain areas, and whether they differ functionally from α7 nAChR homomers. We used α-bungarotoxin to affinity purify α7-containing nAChRs from surgically excised human temporal cortex, and found that α7 subunits co-purify with β2 subunits, indicating the presence of α7β2 nAChRs in the human brain. We validated these results by demonstrating co-purification of β2 from wild-type, but not α7 or β2 knock-out mice. The pharmacology and kinetics of human α7β2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293 cells. Notably, α7β2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with β2 subunits, and that human α7β2 nAChR heteromers respond to nAChR agonists with a unique pharmacology and kinetic profile. α7β2 nAChRs thus represent an alternative mechanism for the reported clinical efficacy of α7 nAChR ligands.

Pharmacological Characterisation of Nicotinic Acetylcholine Receptors Expressed in Human iPSC-Derived Neurons

Neurons derived from human induced pluripotent stem cells (iPSCs) represent a potentially valuable tool for the characterisation of neuronal receptors and ion channels. Previous studies on iPSC-derived neuronal cells have reported the functional characterisation of a variety of receptors and ion channels, including glutamate receptors, γ-aminobutyric acid (GABA) receptors and several voltage-gated ion channels. In the present study we have examined the expression and functional properties of nicotinic acetylcholine receptors (nAChRs) in human iPSC-derived neurons. Gene expression analysis indicated the presence of transcripts encoding several nAChR subunits, with highest levels detected for α3-α7, β1, β2 and β4 subunits (encoded by CHRNA3-CHRNA7, CHRNB1, CHRNB2 and CHRNB4 genes). In addition, similarly high transcript levels were detected for the truncated dupα7 subunit transcript, encoded by the partially duplicated gene CHRFAM7A, which has been associated with psychiatric disorders such as schizophrenia. The functional properties of these nAChRs have been examined by calcium fluorescence and by patch-clamp recordings. The data obtained suggest that the majority of functional nAChRs expressed in these cells have pharmacological properties typical of α7 receptors. Large responses were induced by a selective α7 agonist (compound B), in the presence of the α7-selective positive allosteric modulator (PAM) PNU-120596, which were blocked by the α7-selective antagonist methyllycaconitine (MLA). In addition, a small proportion of the neurons express nAChRs with properties typical of heteromeric (non-α7 containing) nAChR subtypes. These cells therefore represent a great tool to advance our understanding of the properties of native human nAChRs, α7 in particular.

Pharmacological profiling of the TRPV3 channel in recombinant and native assays

Transient receptor potential vanilloid subtype 3 (TRPV3) is implicated in nociception and certain skin conditions. As such, it is an attractive target for pharmaceutical research. Understanding of endogenous TRPV3 function and pharmacology remains elusive as selective compounds and native preparations utilizing higher throughput methodologies are lacking. In this study, we developed medium‐throughput recombinant and native cellular assays to assess the detailed pharmacological profile of human, rat and mouse TRPV3 channels.

Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency – molecular profiling and functional rescue

Mutations in the N-methyl-D-aspartate receptor (NMDAR) gene GRIN2A cause epilepsy-aphasia syndrome (EAS), a spectrum of epileptic, cognitive and language disorders. Using bioinformatic and patient data we shortlisted 10 diverse missense mutations for characterisation. We used high-throughput calcium-flux assays and patch clamp recordings of transiently transfected HEK-293 cells for electrophysiological characterization, and Western blotting and confocal imaging to assay expression and surface trafficking. Mutations P79R, C231Y, G483R and M705V caused a significant reduction in glutamate and glycine agonist potency, whilst D731N was non-responsive. These mutants, along with E714K, also showed significantly decreased total protein levels and trafficking to the cell surface, whilst C436R was not trafficked at all. Crucially this reduced surface expression did not cause the reduced agonist response. We were able to rescue the phenotype of P79R, C231Y, G483R and M705V after treatment with a GluN2A-selective positive allosteric modulator. With our methodology we were not able to identify any functional deficits in mutations I814T, D933N and N976S located between the glutamate-binding domain and C-terminus. We show GRIN2A mutations affect the expression and function of the receptor in different ways. Careful molecular profiling of patients will be essential for future effective personalised treatment options.

An Integrated Approach for Screening and Identification of Positive Allosteric Modulators of N-Methyl-D-Aspartate Receptors:

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an important role in synaptic plasticity and learning and memory formation. Malfunctioning of NMDARs, in particular the reduction in NMDAR activity, is thought to be implicated in major neurological disorders. NMDAR positive allosteric modulators (PAMs) represent potential therapeutic interventions for restoring normal NMDAR function. We report a novel screening approach for identification and characterization of NMDAR-PAMs. The approach combines high-throughput fluorescence imaging with automated electrophysiological recording of glutamate-evoked responses in HEK-293 cells expressing NR1/NR2A NMDAR subunits. Initial high-throughput screening (HTS) of a chemical library containing >810,000 compounds using a calcium flux assay in 1536-well plate format identified a total of 864 NMDAR-PAMs. Concentration response determination in both calcium flux and automated electrophysiological assays found several novel chemical series with EC50 values between 0.49 and 10 µM. A small subset (six series) was selected and analyzed for pharmacological properties, subtype selectivity, mode of action, and activity at native NMDARs. Our approach demonstrates the successful application of HTS functional assays that led to identification of NMDAR-PAMs providing the foundation for further medicinal chemistry work that may lead to novel therapies for treatment of cognitive impairment associated with Alzheimer’s disease and schizophrenia.

The role of Cx36 and Cx43 in 4-aminopyridine-induced rhythmic activity in the spinal nociceptive dorsal horn: an electrophysiological study in vitro

Connexin (Cx) proteins and gap junctions support the formation of neuronal and glial syncytia that are linked to different forms of rhythmic firing and oscillatory activity in the CNS. In this study, quantitative reverse transcription polymerase chain reaction (RT‐qPCR) was used to profile developmental expression of two specific Cx proteins, namely glial Cx43 and neuronal Cx36, in postnatal lumbar spinal cord aged 4, 7, and 14 days. Extracellular electrophysiology was used to determine the contribution of Cx36 and Cx43 to a previously described form of 4‐aminopyridine (4‐AP)‐induced 4–12 Hz rhythmic activity within substantia gelatinosa (SG) of rat neonatal dorsal horn (DH) in vitro. The involvement of Cx36 and Cx43 was probed pharmacologically using quinine, a specific uncoupler of Cx36 and the mimetic peptide blocker Gap 26 which targets Cx43. After establishment of 4–12 Hz rhythmic activity by 4‐AP (25 μmol/L), coapplication of quinine (250 μmol/L) reduced 4‐AP‐induced 4–12 Hz rhythmic activity (P < 0.05). Preincubation of spinal cord slices with Gap 26 (100 μmol/L), compromised the level of 4‐AP‐induced 4–12 Hz rhythmic activity in comparison with control slices preincubated in ACSF alone (P < 0.05). Conversely, the nonselective gap junction “opener” trimethylamine (TMA) enhanced 4–12 Hz rhythmic behavior (P < 0.05), further supporting a role for Cx proteins and gap junctions. These data have defined a physiological role for Cx36 and Cx43 in rhythmic firing in SG, a key nociceptive processing area of DH. The significance of these data in the context of pain and Cx proteins as a future analgesic drug target requires further study.

Synthesis and Pharmacological Characterization of C4β-Amide-Substituted 2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1S,2S,4S,5R,6S)-2-Amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (LY2794193), a Highly Potent and Selective mGlu3 Receptor Agonist

Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu3) receptors, but small molecule tools are lacking. As part of our ongoing efforts to identify potent, selective, and systemically bioavailable agonists for mGlu2 and mGlu3 receptor subtypes, a series of C4β-N-linked variants of (1 S,2 S,5 R,6 S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 1 (LY354740) were prepared and evaluated for both mGlu2 and mGlu3 receptor binding affinity and functional cellular responses. From this investigation we identified (1 S,2 S,4 S,5 R,6 S)-2-amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 8p (LY2794193), a molecule that demonstrates remarkable mGlu3 receptor selectivity. Crystallization of 8p with the amino terminal domain of hmGlu3 revealed critical binding interactions for this ligand with residues adjacent to the glutamate binding site, while pharmacokinetic assessment of 8p combined with its effect in an mGlu2 receptor-dependent behavioral model provides estimates for doses of this compound that would be expected to selectively engage and activate central mGlu3 receptors in vivo.