Homology-guided mutational analysis reveals the functional requirements for antinociceptive specificity of collapsin response mediator protein 2-derived peptides.

BACKGROUND AND PURPOSE: N-type voltage-gated calcium (Cav 2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Cav 2.2 channel antagonists are recommended as first-line treatment for neuropathic pain, calcium-current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Cav 2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15-amino-acid peptide (CBD3), derived from CRMP2, disrupts the functional protein-protein interaction between CRMP2 and Cav 2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential. EXPERIMENTAL APPROACH: Truncated as well as homology-guided mutant versions of CBD3 were generated and assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons, binding between CRMP2 and Cav 2.2 channels, whole-cell voltage clamp electrophysiology and behavioural effects in two models of experimental pain: post-surgical pain and HIV-induced sensory neuropathy induced by the viral glycoprotein 120. KEY RESULTS: The first six amino acids within CBD3 accounted for all in vitro activity and antinociception. Spinal administration of a prototypical peptide (TAT-CBD3-L5M) reversed pain behaviours. Homology-guided mutational analyses of these six amino acids identified at least two residues, Ala1 and Arg4, as being critical for antinociception in two pain models. CONCLUSIONS AND IMPLICATIONS: These results identify an antinociceptive scaffold core in CBD3 that can be used for development of low MW mimetics of CBD3. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Effect of Centruroides antivenom on reversal of methamphetamine-induced hyperkinesis and hyperthermia in rats.

CONTEXT: Methamphetamine (MA) toxicity is a major health concern causing agitation, hyperkinesia, hyperthermia, and even death, affecting 24.7 million people worldwide. It has been observed that MA generates movement disorders in children similar to that of scorpion envenomation. Four cases have been reported where MA intoxication in children were both subjectively and objectively improved as indicated by the reversal of nystagmus and movement disorders following administration of Centruroides antivenom (AV) therapy. OBJECTIVE: Here, we aimed to demonstrate the reversal of MA induced movement disorders and hyperthermia by scorpion AV equine immune F(ab')2 in rats. MATERIALS AND METHODS: Baseline core temperature and locomotor activity in adult male Sprague-Dawley rats (200-220 g) were evaluated prior to acute administration of AV (20 mg/kg, intraperitoneally, i.p.) + MA (10 mg/kg, i.p.) or control. Core body temperature was reassessed 10, 50, and 80 min post injection while locomotor activity was reassessed 20-35 and 60-75 min post injection. RESULTS: At 20-35 min, Saline + MA and BSA + MA groups showed a significant increase in the number of fine events compared to their respective control groups Saline + Saline and BSA + Saline, which indicates an increase in paw movements of animals in situ (p = 0.008, p = 0.006, respectively). In contrast, AV + MA demonstrated a non-significant increase in fine activity compared to the control group AV + Saline). At 60-75 min, the AV + MA treatment group were less likely to engage in locomotor activity indicated by the significant decrease in exploratory events compared to BSA + MA control group (p = 0.041). No significant percent change in core body temperature was observed in the AV + MA treatment group compared to the control groups, AV + Saline and BSA + MA. DISCUSSION: Here, we provide evidence for some aspects of MA-induced hyperkinesia but not hyperthermia reversed by scorpion AV. Further preclinical studies involving adolescent rodents may be necessary to completely mimic the reversal of MA toxicity seen in children in the clinic.

Discovery of amphipathic dynorphin A analogues to inhibit the neuroexcitatory effects of dynorphin A through bradykinin receptors in the spinal cord.

We hypothesized that under chronic pain conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) in the spinal cord to promote hyperalgesia through an excitatory effect, which is opposite to the well-known inhibitory effect of opioid receptors. Considering the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin (BK) and kallidin (KD), this interaction could not be predicted, but it allowed us to discover a potential neuroexcitatory target. Well-known BR ligands, BK, [des-Arg(10), Leu(9)]-kallidin (DALKD), and HOE140 showed different binding profiles at rat brain BRs than that previously reported. These results suggest that neuronal BRs in the rat central nervous system (CNS) may be pharmacologically distinct from those previously defined in non-neuronal tissues. Systematic structure-activity relationship (SAR) study at the rat brain BRs was performed, and as a result, a new key structural feature of Dyn A for BR recognition was identified: amphipathicity. NMR studies of two lead ligands, Dyn A-(4-11) 7 and [des-Arg(7)]-Dyn A-(4-11) 14, which showed the same high binding affinity, confirmed that the Arg residue in position 7, which is known to be crucial for Dyn A's biological activity, is not necessary, and that a type I ß-turn structure at the C-terminal part of both ligands plays an important role in retaining good binding affinities at the BRs. Our lead ligand 14 blocked Dyn A-(2-13) 10-induced hyperalgesic effects and motor impairment in in vivo assays using naïve rats. In a model of peripheral neuropathy, intrathecal (i.th.) administration of ligand 14 reversed thermal hyperalgesia and mechanical hypersensitivity in a dose-dependent manner in nerve-injured rats. Thus, ligand 14 may inhibit abnormal pain states by blocking the neuroexcitatory effects of enhanced levels of Dyn A, which are likely to be mediated by BRs in the spinal cord.

Novel fentanyl-based dual µ/δ-opioid agonists for the treatment of acute and chronic pain.

UNLABELLED: Approximately one third of the adult U.S. population suffers from some type of on-going, chronic pain annually, and many more will have some type of acute pain associated with trauma or surgery. First-line therapies for moderate to severe pain include prescriptions for common mu opioid receptor agonists such as morphine and its various derivatives. The epidemic use, misuse and diversion of prescription opioids have highlighted just one of the adverse effects of mu opioid analgesics. Alternative approaches include novel opioids that target delta or kappa opioid receptors, or compounds that interact with two or more of the opioid receptors. AIMS: Here we report the pharmacology of a newly synthesized bifunctional opioid agonist (RV-Jim-C3) derived from combined structures of fentanyl and enkephalin in rodents. RV-Jim-C3 has high affinity binding to both mu and delta opioid receptors. MAIN METHODS: Mice and rats were used to test RV-Jim-C3 in a tailflick test with and without opioid selective antagonist for antinociception. RV-Jim-C3 was tested for anti-inflammatory and antihypersensitivity effects in a model of formalin-induced flinching and spinal nerve ligation. To rule out motor impairment, rotarod was tested in rats. KEY FINDINGS: RV-Jim-C3 demonstrates potent-efficacious activity in several in vivo pain models including inflammatory pain, antihyperalgesia and antiallodynic with no significant motor impairment. SIGNIFICANCE: This is the first report of a fentanyl-based structure with delta and mu opioid receptor activity that exhibits outstanding antinociceptive efficacy in neuropathic pain, reducing the propensity of unwanted side effects driven by current therapies that are unifunctional mu opioid agonists.