SUMMARY Primary afferents are known to be inhibited by kappa opioid receptor (KOR) signaling. However, the specific types of somatosensory neurons that express KOR remain unclear. Here, using a newly developed KOR-cre knockin allele, viral tracing, single-cell RT-PCR, and ex vivo recordings, we show that KOR is expressed in several populations of primary afferents: a subset of peptidergic sensory neurons, as well as low-threshold mechanoreceptors that form lanceolate or circumferential endings around hair follicles. We find that KOR acts centrally to inhibit excitatory neurotransmission from KOR-cre afferents in laminae I and III, and this effect is likely due to KOR-mediated inhibition of Ca2+ influx, which we observed in sensory neurons from both mouse and human. In the periphery, KOR signaling inhibits neurogenic inflammation, nociceptor sensitization by inflammatory mediators, and pain and itch behaviors. These experiments provide a rationale for the therapeutic use of peripherally restricted KOR agonists.

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 allowed us to discover a potential neuroexcitatory target. Well known BR ligands, BK, 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-Arg7]-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 β-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.

Cholecystokinin (CCK) has been identified as a pronociceptive endogenous peptide which also possesses antiopioid actions. CCK may be upregulated in conditions of chronic pain or during sustained morphine administration resulting in attenuation of opioid-mediated pain relief. These complex interactions between opioids and endogenous CCK receptor systems have suggested the need for a new paradigm in drug design for some states of chronic pain. In these circumstances the rational design of potential drugs for the treatment of these conditions must be based on one ligand for multiple targets. We have designed a single peptide which can interact with δ and μ opioid receptors as agonists and with CCK receptors as antagonists. The ligands were designed based on a model of overlapping pharmacophores of opioid and CCK peptide ligands, which incorporates opioid pharmacophores at the N-terminal and CCK tetrapeptide pharmacophores at the C-terminal of the designed ligands. We measured binding and activities of our bifunctional peptides at opioid and CCK receptors. Compound 11 (Tyr-D-Ala-Gly-D-Trp-NMeNle-Asp-Phe-NH 2 ) demonstrated opioid agonist properties at δ and μ receptors (IC 50 = 63 ± 27 nM and 150 ± 65 nM, respectively in MVD and GPI tissue assays) and high binding affinity at CCK-1 and CCK-2 receptors (K i = 320 and 1.5 nM, respectively). Compound 9 (Tyr-D-Nle-Gly-Trp-Nle-Asp-Phe-NH 2 ) displayed potent agonist activity at δ and μ receptors (IC 50 = 23 ±10 nM and 210 ± 52 nM, respectively in MVD and GPI tissue assays), with a balanced binding affinity for CCK-1 and CCK-2 receptors (K i = 9.6 and 15 nM, respectively). These results provide evidence supporting the concept that opioid and CCK receptors have overlapping pharmacophores required for binding affinity and biological activity and that designing overlapping pharmacophores of two peptides into a single peptide is a valid drug design approach.

CatalyCEST MRI can detect enzyme activity by monitoring the change in chemical exchange with water after a contrast agent is cleaved by an enzyme. Often these molecules use paramagnetic metals and are delivered with an additional non-responsive reference molecule. To improve this approach for molecular imaging, a single diamagnetic agent with enzyme-responsive and enzyme-unresponsive CEST signals was synthesized and characterized. The CEST signal from the aryl amide disappeared after cleavage of a dipeptidyl ligand with cathepsin B, while a salicylic acid moiety was largely unresponsive to enzyme activity. The ratiometric comparison of the two CEST signals from the same agent allowed for concentration independent measurements of enzyme activity. The chemical exchange rate of the salicylic acid moiety was unchanged after enzyme catalysis, which further validated that this moiety was enzyme-unresponsive. The temperature dependence of the chemical exchange rate of the salicylic acid moiety was non-Arrhenius, suggesting a two-step chemical exchange mechanism for salicylic acid. The good detection sensitivity at low saturation power facilitates clinical translation, along with the potentially low toxicity of a non-metallic MRI contrast agent. The modular design of the agent constitutes a platform technology that expands the variety of agents that may be employed by catalyCEST MRI for molecular imaging.

A series of hydrazide-linked bifunctional peptides designed to act as agonists for delta/mu opioid receptors and antagonists for CCK-1/CCK-2 receptors was prepared and tested for binding to both opioid and CCK receptors and in functional assays. SAR studies in the CCK region examined the structural requirements for the side chain groups at positions 1', 2', and 4' and for the N-terminal protecting group, which are related to interactions not only with CCK, but also with opioid receptors. Most peptide ligands that showed high binding affinities (0.1-10 nM) for both delta and mu opioid receptors generally showed lower binding affinities (micromolar range) at CCK-1 and CCK-2 receptors, but were potent CCK receptor antagonists in the GPI/LMMP assay (up to Ke = 6.5 nM). The results indicate that it is reasonable to design chimeric bifunctional peptide ligands for different G-protein coupled receptors in a single molecule.