Sara M. Hall

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-Arg10, Leu9]-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-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.

Modification of amphipathic non-opioid dynorphin A analogues for rat brain bradykinin receptors.

It has been shown that under chronic pain or nerve injury conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) to cause hyperalgesia in the spinal cord. Thus BRs antagonist can modulate hyperalgesia by blocking Dyn As interaction with the BRs in the central nervous system. In our earlier structure-activity relationship (SAR) study, [des-Arg(7)]-Dyn A-(4-11) 13 was discovered as a minimum pharmacophore for rat brain BRs with its antagonist activity (anti-hyperalgesic effect) in in vivo tests using naïve or injured animals. We have pursued further modification on the [des-Arg(7)]-Dyn A analogues and identified a key insight into the pharmacophore of the rat brain BRs: amphipathicity.

Structure–activity relationships of non-opioid [des-Arg7]-dynorphin A analogues for bradykinin receptors

In our earlier studies, bradykinin receptors (BRs) were identified as a potential target for the neuroexcitatory effects of dynorphin A (Dyn A) in the central nervous system (CNS), and [des-Arg(7)]-Dyn A-(4-11) (6) was discovered as a lead ligand to modulate Dyn A-(2-13) induced neuroexcitatory effects in the CNS as an antagonist. In an effort to gain insights into key structural features of the Dyn A for the BRs, we pursued further structure-activity relationships (SAR) study on the [des-Arg(7)]-Dyn A analogs and confirmed that all of the [des-Arg(7)]-Dyn A analogues showed good binding affinities at the BRs.

Discovery of Stable Non-opioid Dynorphin A Analogues Interacting at the Bradykinin Receptors for the Treatment of Neuropathic Pain

Dynorphin A (Dyn A) is a unique endogenous ligand that possesses well-known neuroinhibitory effects via opioid receptors with a preference for the kappa receptor but also neuroexcitatory effects, which cause hyperalgesia. We have shown that the neuroexcitatory effects are mediated through bradykinin (BK) receptors and that intrathecal (i.th.) administration of our lead ligand 1, [des-Arg7]-Dyn A-(4-11), which shows good binding affinity (IC50 = 150 nM) at the BK receptors, blocks Dyn A-induced hyperalgesia in naïve animals and reverses thermal and tactile hypersensitivities in a dose-dependent manner in nerve-injured animals. However, 1 has a serious drawback as a potential drug candidate for the treatment of neuropathic pain because of its susceptibility to enzymatic degradation. In an effort to increase its stability, we modified ligand 1 using non-natural amino acids and found that analogues substituted at or near the N-terminus with a d-isomer retain binding at the receptor and provide a large increase in stability. In particular when Leu5 was modified, with either the d-isomer or N-methylation, there was a large increase in stability (t1/2 = 0.7-160 h in rat plasma) observed. From these studies, we have developed a very stable Dyn A analogue 16, [d-Leu5,des-Arg7]-Dyn A-(4-11), that binds to BK receptors (IC50 = 130 nM) in the same range as ligand 1 and shows good antihyperalgesic effects in both naïve rats and L5/L6 spinal nerve ligation rats.

Dynorphin A analogs for the treatment of chronic neuropathic pain

Chronic pain is one of the most ubiquitous diseases in the world, but treatment is difficult with conventional methods, due to undesirable side effects of treatments and unknown mechanisms of pathological pain states. The endogenous peptide, dynorphin A has long been established as a target for the treatment of pain. Interestingly, this unique peptide has both inhibitory (opioid in nature) and excitatory activities (nonopioid) in the CNS. Both of these effects have been found to play a role in pain and much work has been done to develop therapeutics to enhance the inhibitory effects. Here we will review the dynorphin A compounds that have been designed for the modulation of pain and will discuss where the field stands today.

Various modifications of the amphipathic dynorphin A pharmacophore for rat brain bradykinin receptors.

As a unique endogenous opioid ligand, dynorphin A shows paradoxical neuroexcitatory effects at bradykinin receptors, and the effects are known to be amplified by the upregulation of dynorphin A under chronic pain and inflammatory conditions. In our earlier structure–activity relationship studies, the amphipathic dynorphin A fragment, [Des‐Arg7]‐Dyn A‐(4–11), was identified as a pharmacophore for the bradykinin receptors along with key structural features. Here, further modifications of the pharmacophore showed that the position of a Pro residue is also an important feature because of its role in making (or disrupting) a β‐turn or 310 helix structure which is crucial for receptor recognition.

Cyclic non-opioid dynorphin A analogues for the bradykinin receptors

Nerve injury and inflammation cause up-regulation of an endogenous opioid ligand, dynorphin A (Dyn A), in the spinal cord resulting in hyperalgesia via the interaction with bradykinin receptors (BRs). This is a non-opioid neuroexcitatory effect that cannot be blocked by opioid antagonists. Our systematic structure-activity relationships study on Dyn A identified lead ligands 1 and 4, along with the key structural feature (i.e. amphipathicity) for the BRs. However, the ligands showed very low metabolic stability in plasma (t1/2 <1h) and therefore, in order to improve their metabolic stabilities with retained biological activities, various modifications were performed. Cyclization of ligand 4 afforded a cyclic Dyn A analogue 5 that retained the same range of binding affinity as the linear ligand with improved metabolic stability (t1/2 >5h) and therefore possesses the potential as a pharmacophoric scaffold to be utilized for drug development.

Amphipathic Non-opioid Dynorphin A Analogs to Inhibit Neuroexcitatory Effects at Central Bradykinin Receptors

Nerve injury and inflammation cause up-regulation of dynorphin A (Dyn A, H-Tyr-Gly-Gly-PheLeu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-OH) in the spinal cord, which results in hyperalgesia via the interaction with bradykinin receptors (BRs) [1]. This is a non-opioid neuroexcitatory effect that cannot be blocked by an opioid antagonist, naloxone. On the basis of the fact, systematic structure-activity relationship study on Dyn A was performed to develop BRs antagonists that can block the hyperalgesia. As a result, LYS1044, [des-Arg]-Dyn A-(4-11), was identified as a lead ligand along with key insights into structural features for the BRs recognition (i.e. amphipathicity) [2-4]. Intrathecal administration of the lead ligand reversed thermal hyperalgesia and mechanical hypersensitivity in nerve injured animals and inhibited non-opioid Dyn A-induced motor impairment and hyperalgesia in naive animals. Yet, this ligand showed very low metabolic stabilities in plasma and was completely degraded within 4 hours of incubation (half-life < 1 hour). Therefore, in an effort to improve the metabolic stability and also to enhance the blood brain barrier permeability, various modifications were performed on Dyn A structure. Here we report design and synthesis of cyclic Dyn A analogues and their biological activities.

Structure Activity Relationship of Stable Dynorphin A-(2-13) Analogues Interacting at the Bradykinin-2 Receptor

Neuropathic pain effects 100 million Americans and imposes a significant public health problem. This type of pain results from the dysfunction of the central nervous system (CNS) or the peripheral nervous system (PNS) that can occur in the presence or absence of an initial injury. Some of the current treatments for neuropathic pain involve opioids, non-steroidal anti-inflammatory drugs (NSAIDS), and anticonvulsants, namely gabapentin and pregabalin [1]. Many of these treatments are highly efficacious for acute pain but are not very effective in neuropathic pain and have serious side effects caused by long-term administration. Treatment for this disease is difficult with conventional methods, partly because the mechanism of this disease is not well known. One target for neuropathic pain treatment may be the blockade of Dynorphin A (Dyn A) (Figure 1). Dyn A, a proteolytic fragment derived from prodynorphin, has both inhibitory and excitatory effects in the spinal cord. The inhibitory effects of Dyn A are thought to act primarily through the opioid receptors, with the N-terminal tyrosine being essential for its high affinity and agonist activity. The opioid action of Dyn A is abolished by removing the N-terminal tyrosine, as the des-tyrosyl fragments of Dyn A do not bind to the opioid receptors (IC50 > 10 μM) [2]. However, these fragments are biologically active both in vitro and in vivo, and being neuroexcitatory and neurotoxic, suggest a non-opioid pathway. These des-tyrosyl fragments of Dyn A have been found to bind to the bradykinin 2 receptor (B2R) and cause an influx of calcium [3]. In a chronic pain model, Dyn A was found to be up-regulated and contributes to the maintenance of neuropathic pain [3,4]. Therefore, the development of B2R antagonists can be used to block the agonist actions of Dyn A which may lead to therapeutics for chronic pain.

Structure-Activity Relationship Studies of Dynorphin A Analogs at the Kappa Opioid Receptor

Chronic pain is the most ubiquitous disease with an incidence of 100 million people in the U.S. Opiate therapy is the mainly prescribed treatment for chronic neuropathic pain. However opioids do not address the mechanisms of neuropathic pain and thus have limited efficacy against this type of pain [1]. While opioids may reduce the pain states experienced by the patients, they have adverse effects such as tolerance, addiction, and medication overuse with long-term administration. It has been found that by blocking the κ opioid receptor (KOR) a reduction in tolerance and depressive affective states that can occur with opioid administration [2]. With this in mind we are working towards the development of a KOR selective antagonist with variable duration of action. Dynorphin A (Dyn A, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-AsnGln) is one of three endogenous opioid peptides with high affinities for the μ (MOR), δ (DOR), and κ opioid receptors, with a preference for the KOR. Dyn A mediates an inhibitory effect through the opioid receptors resulting in nociception. Some have studied the importance of consecutive polar residues as is found in Dyn A and found that these were important for binding and function [3]. Dyn A has been extensively studied in the search for KOR ligands. Our approach is different in that our main target is a shorter peptide with an amino acid residue deletion in the middle of the sequence. The deleted residue happens to be one of the key residues determined by studies of the relative importance of the amino acid residues in Dyn A sequence. It was previously believed that residues Arg, Lys, and Lys were the most important residues for binding and potency at the KOR [4]. The importance of these residues was determined by the binding affinity of truncated analogs of Dyn A. In our approach we are completely deleting the amino acid residue and this is very interesting because usually residue deletions would have unfavorable effects to the binding of a peptide due to changes in conformation and charge distributions.