Kimberly M. Lovell

Development of functionally selective, small molecule agonists at kappa opioid receptors

Background: Kappa opioid receptor (KOR) signaling may produce antinociception through G protein or dysphoria through βarrestin pathways. Results: Two highly selective, brain penetrant agonist scaffolds bias KOR signaling toward G protein coupling and produce antinociception in mice. Conclusion: Described are first-in-class small molecule agonists that bias KOR signaling through G proteins. Significance: Functionally selective KOR agonists can now be used in vivo. The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as a means to modulate pain perception, stress responses, and affective reward states. Therefore, the KOR has become a prominent drug discovery target toward treating pain, depression, and drug addiction. Agonists at KOR can promote G protein coupling and βarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAPK activation. It has been suggested that the physiological effects of KOR activation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through βarrestin2 recruitment. Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased toward G protein coupling and away from βarrestin2 recruitment. Here, we describe two classes of biased KOR agonists that potently activate G protein coupling but weakly recruit βarrestin2. These potent and functionally selective small molecule compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo.

Fragment-based screening by X-ray crystallography, MS and isothermal titration calorimetry to identify PNMT (phenylethanolamine N-methyltransferase) inhibitors.

CNS (central nervous system) adrenaline (epinephrine) is implicated in a wide range of physiological and pathological conditions. PNMT (phenylethanolamine N-methyltransferase) catalyses the final step in the biosynthesis of adrenaline, the conversion of noradrenaline (norepinephrine) to adrenaline by methylation. To help elucidate the role of CNS adrenaline, and to develop potential drug leads, potent, selective and CNS-active inhibitors are required. The fragment screening approach has advantages over other lead discovery methods including high hit rates, more efficient hits and the ability to sample chemical diversity more easily. In the present study we applied fragment-based screening approaches to the enzyme PNMT. We used crystallography as the primary screen and identified 12 hits from a small commercial library of 384 drug-like fragments. The hits include nine chemicals with two fused rings and three single-ring chemical systems. Eight of the hits come from three chemical classes: benzimidazoles (a known class of PNMT inhibitor), purines and quinolines. Nine of the hits have measurable binding affinities (~5-700 μM) as determined by isothermal titration calorimetry and all nine have ligand efficiencies of 0.39 kcal/mol per heavy atom or better (1 kcal≈4.184 kJ). We synthesized five elaborated benzimidazole compounds and characterized their binding to PNMT, showing for the first time how this class of inhibitors interact with the noradrenaline-binding site. Finally, we performed a pilot study with PNMT for fragment-based screening by MS showing that this approach could be used as a fast and efficient first-pass screening method prior to characterization of binding mode and affinity of hits.

Bias Factor and Therapeutic Window Correlate to Predict Safer Opioid Analgesics

Biased agonism has been proposed as a means to separate desirable and adverse drug responses downstream of G protein-coupled receptor (GPCR) targets. Herein, we describe structural features of a series of mu-opioid-receptor (MOR)-selective agonists that preferentially activate receptors to couple to G proteins or to recruit βarrestin proteins. By comparing relative bias for MOR-mediated signaling in each pathway, we demonstrate a strong correlation between the respiratory suppression/antinociception therapeutic window in a series of compounds spanning a wide range of signaling bias. We find that βarrestin-biased compounds, such as fentanyl, are more likely to induce respiratory suppression at weak analgesic doses, while G protein signaling bias broadens the therapeutic window, allowing for antinociception in the absence of respiratory suppression.

Structure–Activity Relationship Studies of Functionally Selective Kappa Opioid Receptor Agonists that Modulate ERK 1/2 Phosphorylation While Preserving G Protein Over βArrestin2 Signaling Bias

Kappa opioid receptor (KOR) modulation is a promising target for drug discovery efforts due to KOR involvement in pain, depression, and addiction behaviors. We recently reported a new class of triazole KOR agonists that displays significant bias toward G protein signaling over βarrestin2 recruitment; interestingly, these compounds also induce less activation of ERK1/2 map kinases than the balanced agonist, U69,593. We have identified structure-activity relationships around the triazole scaffold that allows for decreasing the bias for G protein signaling over ERK1/2 activation while maintaining the bias for G protein signaling over βarrestin2 recruitment. The development of novel compounds, with different downstream signaling outcomes, independent of G protein/βarrestin2 bias, provides a more diverse pharmacological toolset for use in defining complex KOR signaling and elucidating the significance of KOR-mediated signaling.

Potential Drug Abuse Therapeutics Derived from the Hallucinogenic Natural Product Salvinorin A.

Previous structure-activity relationship studies of salvinorin A have shown that modification of the acetate functionality off the C-2 position to a methoxy methyl or methoxy ethyl ether moiety leads to increased potency at KOP receptors. However, the reason for this increase remains unclear. Here we report our efforts towards the synthesis and evaluation of C-2 constrained analogs of salvinorin A. These analogs were evaluated at opioid receptors in radioligand binding experiments as well as in the GTP-γ-S functional assay. One compound, 5, was found to have affinity and potency at κ opioid (KOP) receptors comparable to salvinorin A. In further studies, 5 was found to attenuate cocaine-induced drug seeking behavior in rats comparably to salvinorin A. This finding represents the first example of a salvinorin A analog that has demonstrated anti-addictive capabilities.

Semisynthetic Neoclerodanes as Kappa Opioid Receptor Probes

Modification of the furan ring of salvinorin A (1), the main active component of Salvia divinorum, has resulted in novel neoclerodane diterpenes with opioid receptor affinity and activity. Conversion of the furan ring to an aldehyde at the C-12 position (5) has allowed for the synthesis of analogues with new carbon-carbon bonds at that position. Previous methods for forming these bonds, such as Grignard and Stille conditions, have met with limited success. We report a palladium catalyzed Liebeskind-Srogl cross-coupling reaction of a thioester and a boronic acid that occurs at neutral pH and ambient temperature to produce ketone analogs at C-12. To the best of our knowledge, this is the first reported usage of the Liebeskind-Srogl reaction to diversify a natural product scaffold. We also describe a one-step protocol for the conversion of 1 to 12-epi-1 (3) through microwave irradiation. Previously, this synthetically challenging process has required multiple steps. Additionally, we report in this study that alkene 9 and aromatic analogues 12, 19, 23, 25, and 26 were discovered to retain affinity and selectivity at kappa opioid receptors (KOP). Finally, we report that the furan-2-yl analog of 1 (31) has similar affinity to 1. Collectively, these findings suggest that different aromatic groups appended directly to the decalin core may be well tolerated by KOP receptors, and may generate further ligands with affinity and activity at KOP receptors.

Synthetic Studies of Neoclerodane Diterpenes from Salvia Divinorum: Role of the Furan in Affinity for Opioid Receptors

Further synthetic modification of the furan ring of salvinorin A (1), the major active component of Salvia divinorum, has resulted in novel neoclerodane diterpenes with opioid receptor affinity and activity. A computational study has predicted 1 to be a reproductive toxicant in mammals and is suggestive that use of 1 may be associated with adverse effects. We report in this study that piperidine 21 and thiomorpholine 23 have been identified as selective partial agonists at kappa opioid receptors. This indicates that additional structural modifications of 1 may provide ligands with good selectivity for opioid receptors but with reduced potential for toxicity.

Behavioral Effects and Central Nervous System Levels of the Broadly Available κ-Agonist Hallucinogen Salvinorin A Are Affected by P-Glycoprotein Modulation In Vivo

Active blood-brain barrier mechanisms, such as the major efflux transporter P-glycoprotein (mdr1), modulate the in vivo/central nervous system (CNS) effects of many pharmacological agents, whether they are used for nonmedical reasons or in pharmacotherapy. The powerful, widely available hallucinogen salvinorin A (from the plant Salvia divinorum) is a high-efficacy, selective κ-opioid agonist and displays fast-onset behavioral effects (e.g., within 1 min of administration) and relatively short duration of action. In vitro studies suggest that salvinorin A may be a P-glycoprotein substrate; thus, the functional status of P-glycoprotein may influence the behavioral effects of salvinorin A or its residence in CNS after parenteral administration. We therefore studied whether a competing P-glycoprotein substrate (the clinically available agent loperamide; 0.032–0.32 mg/kg) or a selective P-glycoprotein blocker, tariquidar (0.32–3.2 mg/kg) could enhance unconditioned behavioral effects (ptosis and facial relaxation, known to be caused by κ-agonists in nonhuman primates) of salvinorin A, as well as its entry and residence in the CNS, as measured by cerebrospinal fluid sampling. Pretreatment with either loperamide or tariquidar dose-dependently enhanced salvinorin A-induced ptosis, but not facial relaxation. In a control study, loperamide and tariquidar were inactive when given as a pretreatment to ((+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U69,593), a κ-agonist known to be a very poor P-glycoprotein substrate. Furthermore, pretreatment with tariquidar (3.2 mg/kg) also enhanced peak levels of salvinorin A in cerebrospinal fluid after intravenous administration. These are the first studies in vivo showing the sensitivity of salvinorin A effects to modulation by the P-glycoprotein transporter, a major functional component of the blood-brain barrier.

Synthesis of Neoclerodane Diterpenes and Their Pharmacological Effects

Salvinorin A is a neoclerodane diterpene that has been shown to be an agonist at kappa opioid receptors. Its unique structure makes it an attractive target for synthetic organic chemists due to its seven chiral centers and diterpene scaffold. This molecule is also interesting to pharmacologists because it is a non-serotonergic hallucinogen, and the first opioid ligand discovered that lacks a basic nitrogen. There have been several total synthesis approaches to salvinorin A, and these will be detailed within this chapter. Additionally, research efforts have concentrated on structure modification of the salvinorin A scaffold through semi-synthetic methods. Most modifications have focused on the manipulation of the acetate at C-2 and the furan ring. However, chemistry has also been developed to generate analogs at the C-1 ketone, the C-4 methyl ester, and the C-17 lactone. The synthetic methodologies developed for the salvinorin A scaffold will be described, as well as specific analogs with interesting biological activities.

Potency enhancement of the κ-opioid receptor antagonist probe ML140 through sulfonamide constraint utilizing a tetrahydroisoquinoline motif

Optimization of the sulfonamide-based kappa opioid receptor (KOR) antagonist probe molecule ML140 through constraint of the sulfonamide nitrogen within a tetrahydroisoquinoline moiety afforded a marked increase in potency. This strategy, when combined with additional structure-activity relationship exploration, has led to a compound only six-fold less potent than norBNI, a widely utilized KOR antagonist tool compound, but significantly more synthetically accessible. The new optimized probe is suitably potent for use as an in vivo tool to investigate the therapeutic potential of KOR antagonists.