Suneeta Tumati

A Structure–Activity Relationship Study and Combinatorial Synthetic Approach of C-Terminal Modified Bifunctional Peptides That Are δ/μ Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists

A series of bifunctional peptides with opioid agonist and substance P antagonist bioactivities were designed with the concept of overlapping pharmacophores. In this concept, the bifunctional peptides were expected to interact with each receptor separately in the spinal dorsal horn where both the opioid receptors and the NK1 receptors were found to be expressed, to show an enhanced analgesic effect, no opioid-induced tolerance, and to provide better compliance than coadministration of two drugs. Compounds were synthesized using a two-step combinatorial method for C-terminal modification. In the method, the protected C-terminal-free carboxyl peptide, Boc-Tyr( tBu)- d-Ala-Gly Phe-Pro-Leu-Trp(Boc)-OH, was synthesized as a shared intermediate using Fmoc solid phase chemistry on a 2-chlorotrityl resin. This intermediate was esterified or amidated in solution phase. The structure-activity relationships (SAR) showed that the C-terminus acted as not only a critical pharmacophore for the substance P antagonist activities, but as an address region for the opioid agonist pharmacophore that is structurally distant from the C-terminal. Among the peptides, H-Tyr- d -Ala-Gly-Phe-Pro-Leu-Trp-NH-Bzl ( 3) demonstrated high binding affinities at both delta and mu receptors ( K i = 10 and 0.65 nM, respectively) with efficient agonist functional activity in the mouse isolated vas deferens (MVD) and guinea pig isolated ileum (GPI) assays (IC 50 = 50 and 13 nM, respectively). Compound 3 also showed a good antagonist activity in the GPI assay with substance P stimulation ( K e = 26 nM) and good affinity for the hNK1 receptor ( K i = 14 nM). Consequently, compound 3 is expected to be a promising and novel type of analgesic with bifunctional activities.

Sustained morphine treatment augments basal CGRP release from cultured primary sensory neurons in a Raf-1 dependent manner.

Recent studies suggest that sustained morphine-mediated paradoxical pain may play an important role in the development of analgesic tolerance. The intracellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) release are not fully clarified. Cyclic AMP (cAMP)-dependent protein kinase (PKA) plays an important role in the modulation of presynaptic neurotransmitter release. Moreover, we have shown earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). Therefore, in the present study we examined the role of Raf-1 in sustained morphine-mediated regulation of cAMP formation and basal CGRP release in vitro, in cultured neonatal rat dorsal root ganglion (DRG) neurons. We found that sustained morphine treatment significantly augments intracellular cAMP production as well as basal CGRP release from cultured neonatal rat DRG neurons. The selective PKA inhibitor, H-89, attenuates the sustained morphine-mediated augmentation of basal CGRP release, indicating that the cAMP/PKA pathway plays an important role in regulation of CGRP release from sensory neurons. Since our present data also demonstrated that selective Raf-1 inhibitor, GW 5074, attenuated both the cAMP overshoot and the augmentation of CGRP release mediated by sustained morphine in neonatal rat DRG neurons, we suggest that Raf-1-mediated sensitization of the intracellular cAMP formation may play an important role in sustained morphine-mediated augmentation of spinal pain neurotransmitter release.

Sustained Morphine Treatment Augments Capsaicin-Evoked Calcitonin Gene-Related Peptide Release from Primary Sensory Neurons in a Protein Kinase A- and Raf-1-Dependent Manner

Studies have shown that long-term (5α,6α)-7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol (morphine) treatment increases the sensitivity to painful heat stimuli (thermal hyperalgesia). The cellular adaptations contributing to sustained morphine-mediated pain sensitization are not fully understood. It was shown previously (J Neurosci 22:6747–6755, 2002) that sustained morphine exposure augments pain neurotransmitter [such as calcitonin gene-related peptide (CGRP)] release in the dorsal horn of the spinal cord in response to the heat-sensing transient receptor potential vanilloid 1 receptor agonist 8-methyl-N-vanillyl-6-nonenamide (capsaicin). In the present study, we demonstrate that sustained morphine-mediated augmentation of CGRP release from isolated primary sensory dorsal root ganglion neurons is dependent on protein kinase A and Raf-1 kinase. Our data indicate that, in addition to neural system adaptations, sustained opioid agonist treatment also produces intracellular compensatory adaptations in primary sensory neurons, leading to augmentation of evoked pain neurotransmitter release from these cells.

Repeated morphine treatment-mediated hyperalgesia, allodynia and spinal glial activation are blocked by co-administration of a selective cannabinoid receptor type-2 agonist

Spinal glial activation has been implicated in sustained morphine-mediated paradoxical pain sensitization. Since activation of glial CB2 cannabinoid receptors attenuates spinal glial activation in neuropathies, we hypothesized that CB2 agonists may also attenuate sustained morphine-mediated spinal glial activation and pain sensitization. Our data indicate that co-administration of a CB2-selective agonist (AM 1241) attenuates morphine (intraperitoneal; twice daily; 6 days)-mediated thermal hyperalgesia and tactile allodynia in rats. A CB2 (AM 630) but not a CB1 (AM 251) antagonist mitigated this effect. AM 1241 co-treatment also attenuated spinal astrocyte and microglial marker and pro-inflammatory mediator (IL-1β, TNFα) immunoreactivities in morphine-treated rats, suggesting that CB2 agonists may be useful to prevent the neuroinflammatory consequences of sustained morphine treatment.

Intrathecal PKA-selective siRNA treatment blocks sustained morphine-mediated pain sensitization and antinociceptive tolerance in rats

Sustained morphine treatment has been shown to produce paradoxical pain sensitization (opioid-induced hyperalgesia) and also causes increase in spinal pain neurotransmitter, such as calcitonin gene related peptide (CGRP), concentration in experimental animals. Studies have also shown that cyclic adenosine-monophosphate (cAMP)-dependent protein kinase (PKA) plays a major role in the regulation of presynaptic neurotransmitter (such as CGRP and substance P) synthesis and release. We have previously shown that in cultured primary sensory dorsal root ganglion (DRG) neurons sustained in vitro opioid agonist treatment upregulates cAMP levels (adenylyl cyclase (AC) superactivation) and augments basal and capsaicin evoked CGRP release in a PKA dependent manner. In the present study, we investigated the in vivo role of PKA in sustained morphine-mediated pain sensitization. Our data indicate that selective knock-down of spinal PKA activity by intrathecal (i.th.) pretreatment of rats with a PKA-selective small interference RNA (siRNA) mixture significantly attenuates sustained morphine-mediated augmentation of spinal CGRP immunoreactivity, thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. The present findings indicate that sustained morphine-mediated activation of spinal cAMP/PKA-dependent signaling may play an important role in opioid induced hyperalgesia.

Sustained morphine treatment augments prostaglandin E2-evoked calcitonin gene-related peptide release from primary sensory neurons in a PKA-dependent manner.

Tissue damage leads to pain sensitization due to peripheral and central release of excitatory mediators such as prostaglandin E₂ (PGE₂). PGE₂ sensitizes spinal pain neurotransmitter such as calcitonin gene-related peptide (CGRP) release via activation of cyclic AMP (cAMP)/protein kinase A (PKA)-dependent signaling mechanisms. Our previous data demonstrate that sustained morphine pretreatment sensitizes adenylyl cyclase(s) (AC) toward the direct stimulator, forskolin, in cultured primary sensory neurons (AC superactivation). In the present work we investigated the hypothesis that morphine pretreatment also sensitizes ACs toward Gs-protein-coupled excitatory modulators (such as PGE₂), leading to augmented PKA-dependent CGRP release from PGE₂-stimulated primary sensory dorsal root ganglion (DRG) neurons. Our results show that sustained morphine treatment potentiated PGE₂-mediated cAMP formation and augmented PGE₂-evoked CGRP release from cultured primary sensory neurons in a PKA-dependent manner. Our data suggest that attenuation of AC superactivation in primary sensory neurons may prevent the development of opioid-induced hyperalgesia.

Intrathecal Raf-1-selective siRNA attenuates sustained morphine-mediated thermal hyperalgesia

Studies have demonstrated that long-term opioid treatment leads to an increased sensitivity to painful (hyperalgesia) or normally innocuous (allodynia) stimuli. The molecular mechanisms that lead to paradoxical pain sensitization upon chronic opioid treatment are not completely understood. Enhanced excitatory pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) release in the dorsal horn of the spinal cord may play a role in sustained morphine-mediated paradoxical pain. Recently we have demonstrated that inhibition of Raf-1 attenuates sustained morphine treatment-mediated augmentation of CGRP release in vitro, in cultured primary sensory neurons. In the present study, we show that knockdown of spinal Raf-1 levels in vivo by intrathecal administration of Raf-1-specific siRNA attenuates sustained morphine-mediated thermal hyperalgesia in rats.

Functional Selectivity in Cannabinoid Signaling

Cannabinoid (CB) agonists exhibit numerous potentially useful pharmacological properties, but unwanted side effects limit their use in clinical practice. Thus, novel strategies are needed to identify potential CB pharmaceuticals with fewer side effects. Activated CB receptors initiate multiple parallel intracellular signal transduction cascades. In the present paper we will review experimental data indicating that structurally different classes of CB agonists may exhibit selectivity toward individual subsets of intracellular signaling pathways. In support of this, recent findings indicate that chemically distinct classes of CB agonists frequently differ in their rank order of potency to produce analgesia versus other central nervous system effects in vivo. Structurally different agonists were also found to differ in their abilities to activate individual G protein types in vitro. Since it was suggested earlier that structurally distinct CB agonists may interact differently with the CB receptors, it has been hypothesized that different classes of cannabinoid agonists may stabilize unique active CB receptor conformations, leading to functional selectivity in CB receptor signaling. In order to obtain a direct proof for this hypothesis, we recently employed a highly sensitive biophysical method, plasmon-waveguide resonance (PWR) spectroscopy. PWR experiments have provided a direct proof that structurally different CB agonists produce qualitatively distinct changes in the shape and/or membrane orientation of the CB1 receptors, leading to functional selectivity in G protein activation. We expect that by identification of CB agonists that selectively activate preferred intracellular signaling pathways novel pharmacological lead structures can be identified for the design of improved CB analgesics with fewer side effects.

Sustained morphine‐mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf‐1‐selective siRNA

BACKGROUND AND PURPOSE Long‐term morphine treatment enhances pain neurotransmitter [such as calcitonin gene‐related peptide (CGRP)] levels in the spinal cord. It has been suggested previously that increased spinal CGRP may contribute to sustained morphine‐mediated paradoxical pain sensitization and antinociceptive tolerance. Previous in vitro studies from our group indicated that Raf‐1 kinase‐mediated adenylyl cyclase superactivation played a crucial role in sustained morphine‐mediated augmentation of basal and evoked CGRP release from cultured primary sensory neurons. The present study was aimed to evaluate the physiological significance of this molecular mechanism in vivo, in rats.

Plasmon-waveguide resonance studies of ligand binding to integral proteins in membrane fragments derived from bacterial and mammalian cells

A procedure has been developed for directly depositing membrane fragments derived from bacterial cells (chromatophores from Rhodopseudomonas sphaeroides) and mammalian cells (mu-opioid receptor- and MC4 receptor-transfected human embryonic kidney (HEK) cells and rat trigeminal ganglion cells) on the silica surface of a plasmon-waveguide resonance (PWR) spectrometer. Binding of ligands (cytochrome c(2) for the chromatophores, the peptide agonists DAMGO and melanotan-II that are specific for the mu-opioid and MC4 receptors, and two nonpeptide agonists that are specific for the CB1 receptor) to these membrane fragments has been observed and characterized with high sensitivity using PWR spectral shifts. The K(D) values obtained are in excellent agreement with conventional pharmacological assays and with prior PWR studies using purified receptors inserted into deposited lipid bilayer membranes. These studies provide a new tool for obtaining useful biological information about receptor-mediated processes in real biological membranes.