Pharmacology/Drug Development

A decarboxylated form of L-arginine, agmatine, preferentially antagonizes NMDArs containing Glun2B subunits within the spinal cord and lacks motor side effects commonly associated with non-subunit-selective NMDAr antagonism, namely sedation and motor impairment. Spinally delivered agmatine has been previously shown to reduce the development of tactile hypersensitivity arising from spinal nerve ligation. The present study interrogated the dependence of agmatine's alleviation of neuropathic pain (spared nerve injury (SNI) model) on GluN2B-containing NMDArs. SNI-induced hypersensitivity was induced in mice with significant reduction of levels of spinal GluN2B subunit of the NMDAr and their floxed controls. Agmatine reduced development of SNI-induced tactile hypersensitivity in controls but had no effect in subjects with reduced levels of GluN2B subunits. Ifenprodil, a known GluN2B-subunit-selective antagonist, similarly reduced tactile hypersensitivity in controls but not in the GluN2B-deficient mice. In contrast, MK-801, an NMDA receptor channel blocker, reduced hypersensitivity in both control and GluN2B-deficient mice, consistent with a pharmacological pattern expected from a NMDAr antagonist that does not have preference for GluN2B subtypes. Additionally, we observed that spinally delivered agmatine, ifenprodil and MK-801 inhibited nociceptive behaviors following intrathecal delivery of NMDA in control mice. By contrast, in GluN2B-deficient mice, MK-801 reduced NMDA-evoked nociceptive behaviors, but agmatine had a blunted effect and ifenprodil had no effect. These results demonstrate that agmatine requires the GluN2B subunit of the NMDA receptor for inhibitory pharmacological actions in pre-clinical models of NMDA receptor-dependent hypersensitivity.

The cone snails (family Conidae) are the best known and most intensively studied venomous marine gastropods. However, of the total biodiversity of venomous marine mollusks (superfamily Conoidea, >20,000 species), cone snails comprise a minor fraction. The venoms of the family Drilliidae, a highly diversified family in Conoidea, have not previously been investigated. In this report, we provide the first biochemical characterization of a component in a Drilliidae venom and define a gene superfamily of venom peptides. A bioactive peptide, cdg14a, was purified from the venom of Fedosov and Puillandre, 2020. The peptide is small (23 amino acids), disulfide-rich (4 cysteine residues) and belongs to the J-like drillipeptide gene superfamily. Other members of this superfamily share a conserved signal sequence and the same arrangement of cysteine residues in their predicted mature peptide sequences. The cdg14a peptide was chemically synthesized in its bioactive form. It elicited scratching and hyperactivity, followed by a paw-thumping phenotype in mice. Using the Constellation Pharmacology platform, the cdg14a drillipeptide was shown to cause increased excitability in a majority of non-peptidergic nociceptors, but did not affect other subclasses of dorsal root ganglion (DRG) neurons. This suggests that the cdg14a drillipeptide may be blocking a specific molecular isoform of potassium channels. The potency and selectivity of this biochemically characterized drillipeptide suggest that the venoms of the Drilliidae are a rich source of novel and selective ligands for ion channels and other important signaling molecules in the nervous system.

Selective activation of the δ-opioid receptor (DOP) has great potential for the treatment of chronic pain, benefitting from ancillary anxiolytic and antidepressant-like effects. Moreover, DOP agonists show reduced adverse effects as compared to μ-opioid receptor (MOP) agonists that are in the spotlight of the current "opioid crisis." Here, we report the first crystal structures of the DOP in an activated state, in complex with two relevant and structurally diverse agonists: the potent opioid agonist peptide KGCHM07 and the small-molecule agonist DPI-287 at 2.8 and 3.3 Å resolution, respectively. Our study identifies key determinants for agonist recognition, receptor activation, and DOP selectivity, revealing crucial differences between both agonist scaffolds. Our findings provide the first investigation into atomic-scale agonist binding at the DOP, supported by site-directed mutagenesis and pharmacological characterization. These structures will underpin the future structure-based development of DOP agonists for an improved pain treatment with fewer adverse effects.