Solving an Old Puzzle: Elucidation and Evaluation of the Binding Mode of Salvinorin A at the Kappa Opioid Receptor

The natural product Salvinorin A (SalA) was the first nitrogen-lacking agonist discovered for the opioid receptors and exhibits high selectivity for the kappa opioid receptor (KOR) turning SalA into a promising analgesic to overcome the current opioid crisis. Since SalA’s suffers from poor pharmacokinetic properties, particularly the absence of gastrointestinal bioavailability, fast metabolic inactivation, and subsequent short duration of action, the rational design of new tailored analogs with improved clinical usability is highly desired. Despite being known for decades, the binding mode of SalA within the KOR remains elusive as several conflicting binding modes of SalA were proposed hindering the rational design of new analgesics. In this study, we rationally determined the binding mode of SalA to the active state KOR by in silico experiments (docking, molecular dynamics simulations, dynophores) in the context of all available mutagenesis studies and structure-activity relationship (SAR) data. To the best of our knowledge, this is the first comprehensive evaluation of SalA’s binding mode since the determination of the active state KOR crystal structure. SalA binds above the morphinan binding site with its furan pointing toward the intracellular core while the C2-acetoxy group is oriented toward the extracellular loop 2 (ECL2). SalA is solely stabilized within the binding pocket by hydrogen bonds (C210ECL2, Y3127.35, Y3137.36) and hydrophobic contacts (V1182.63, I1393.33, I2946.55, I3167.39). With the disruption of this interaction pattern or the establishment of additional interactions within the binding site, we were able to rationalize the experimental data for selected analogs. We surmise the C2-substituent interactions as important for SalA and its analogs to be experimentally active, albeit with moderate frequency within MD simulations of SalA. We further identified the non-conserved residues 2.63, 7.35, and 7.36 responsible for the KOR subtype selectivity of SalA. We are confident that the elucidation of the SalA binding mode will promote the understanding of KOR activation and facilitate the development of novel analgesics that are urgently needed

Exploration of Salvinorin A for the Development of Pain and Addiction Therapies

In the search for effective methods to mitigate the increasing rates of abuse and addiction of illicit substances, a variety of neurological pathways have been explored. Towards this goal of reducing drug abuse and ultimately overdose-related deaths, two avenues of research have emerged: 1) a preventative approach, the development of pain-relieving medications without the abuse and addiction liabilities associated with current therapies, and 2) a responsive approach, the development of medications for people suffering from drug abuse and addiction. The natural product salvinorin A can be manipulated towards both of these research avenues through the development of opioid receptor (MOR) agonists for treating pain with reduced abuse liabilities as well as through the development of opioid receptor (KOR) agonists with improved pharmacokinetic properties towards the development of therapies that attenuate relapse and withdrawal. Previous structure-activity relationship (SAR) studies of salvinorin A identified that replacing the C2-acetate with a C2-benzoate results in a compound that is 4-fold selective for MORs over KORs. In an effort to increase this selectivity, to allow for probing the physiological effects induced upon activation of MORs with this non-morphine scaffold, a potent and selective MOR agonist kurkinorin (MOR EC50 = 1.2 ± 0.6 nM, and KOR > 10,000nM) was identified. Upon in vivo evaluation, kurkinorin was determined to elicit centrally-mediated antinociception with similar potency to morphine and a reduced tolerance, sedation, and reward profile in comparison to morphine. Through a SAR campaign, a variety of kurkinorin analogues were synthesized and evaluated in vitro for their ability to activate G-proteins and recruit β-arrestin-2 upon MOR activation. Through these studies, compounds that are more potent than kurkinorin at MORs, compounds that are biased towards β-arrestin-2 recruitment, and compounds that are biased towards G-protein activation have been identified. Salvinorin A suffers from poor pharmacokinetic properties, including low water solubility and rapid metabolism. In an effort to address this issue of water solubility, we sought to identify a point on the molecule through which its water solubility could be modified without sacrificing KOR activity. Previous studies indicated that salvinorin A’s lactone functionality was not necessary for KOR activity; therefore, the lactone was modified to further explore its necessity and tolerance to modification. Analogues that varied in chain length, stereochemistry, and polarity at the lactone position were synthesized and evaluated for their ability to activate KORs. Overall, small linear moieties were well-tolerated, while bulkier groups were not. Salvinorin A analogues that are potent KOR agonists with polar, ionizable moieties in the C17-position have been identified, and the lactone position has been validated as a position on the molecule through which the pharmacokinetic properties can be manipulated without significant loss of KOR activity. Salvinorin A has a very short half-life in humans ( 10,000nM) was identified. Upon in vivo evaluation, kurkinorin was determined to elicit centrally-mediated antinociception with similar potency to morphine and a reduced tolerance, sedation, and reward profile in comparison to morphine. Through a SAR campaign, a variety of kurkinorin analogues were synthesized and evaluated in vitro for their ability to activate G-proteins and recruit β-arrestin-2 upon MOR activation. Through these studies, compounds that are more potent than kurkinorin at MORs, compounds that are biased towards β-arrestin-2 recruitment, and compounds that are biased towards G-protein activation have been identified. Salvinorin A suffers from poor pharmacokinetic properties, including low water solubility and rapid metabolism. In an effort to address this issue of water solubility, we sought to identify a point on the molecule through which its water solubility could be modified without sacrificing KOR activity. Previous studies indicated that salvinorin A’s lactone functionality was not necessary for KOR activity; therefore, the lactone was modified to further explore its necessity and tolerance to modification. Analogues that varied in chain length, stereochemistry, and polarity at the lactone position were synthesized and evaluated for their ability to activate KORs. Overall, small linear moieties were well-tolerated, while bulkier groups were not. Salvinorin A analogues that are potent KOR agonists with polar, ionizable moieties in the C17-position have been identified, and the lactone position has been validated as a position on the molecule through which the pharmacokinetic properties can be manipulated without significant loss of KOR activity. Salvinorin A has a very short half-life in humans (<15 minutes upon inhalation). SAR work in the past has focused on developing analogues with reduced metabolic liabilities, specifically through replacement of the acetate moiety, while maintaining KOR activity. However, the metabolism of many of these compounds had not been directly compared, to one another or to salvinorin A. Therefore, we developed a method to analyze the metabolic profiles of salvinorin A and its analogues in liver microsomes. Through screening salvinorin A and its analogues, several modifications that increase metabolic stability in comparison with salvinorin A have been identified

Investigation of Natural Product Scaffolds for the Development of Opioid Receptor Ligands

Kappa opioid (KOP) receptors have been suggested as an alternative target to the mu opioid (MOP) receptor for the treatment of pain because KOP activation is associated with fewer negative side-effects (respiratory depression, constipation, tolerance, and dependence). The KOP receptor has also been implicated in several abuse-related effects in the central nervous system (CNS). KOP ligands have been investigated as pharmacotherapies for drug abuse; KOP agonists have been shown to modulate dopamine concentrations in the CNS as well as attenuate the self-administration of cocaine in a variety of species, and KOP antagonists have potential in the treatment of relapse. One drawback of current opioid ligand investigation is that many compounds are based on the morphine scaffold and thus have similar properties, both positive and negative, to the parent molecule. Thus there is increasing need to discover new chemical scaffolds with opioid receptor activity. The flavonoid class of natural products has been identified as a potential source of novel opioid ligands. In particular, dioclein (86) and dioflorin (87) have been reported to have an antinociceptive effect in rodent models of pain, although there has been no in vitro pharmacological evaluation to date. Dioclein and several simplified analogs of dioflorin were synthesized in order to develop structure activity relationships (SAR) for the flavonoid scaffold at opioid receptors. The analogs were pharmacologically evaluated in several cell-based assays (radioligand binding, fluorescent calcium mobilization, and luminescent PathHunterTM beta-arrestin) and found to be inactive at both opioid and cannabinoid receptors. The novel KOP receptor agonist and neoclerodane diterpene salvinorin A was also investigated. Salvinorin A is the main active component of the hallucinogenic plant Salvia divinorum and is the first opioid ligand reported that lacks a basic nitrogen atom in the structure. Ether analogs at the C-2 position of salvinorin A have been reported to have improved affinity and potency over the parent molecule. As alkyl chain ethers have a high degree of flexibility and the oxygen atom may allow for extra hydrogen bonding interactions in the receptor, modifications were made at the C-2 position in order to develop analogs to elucidate the molecular basis for this improved affinity and potency. Tetrahydropyranyl ether 220, ether 223a, and methyltetrahydropyranyl ether 231 were found to have similar KOP affinity and potency to salvinorin A in radioligand binding, [35S]GTP-gamma-S functional, and fluorescent calcium mobilization assays. Tetrahydropyranyl ether 220 was further evaluated for its effects on the cocaine-primed reinstatement of extinguished cocaine self-administration in rats; 220 (1 mg/kg) was found to attenuate cocaine self-administration comparably to salvinorin A (0.3 mg/kg), previously reported to be effective in this animal model. This represents the first report of a salvinorin A derivative with demonstrated anti-addictive capability

IUPHAR themed review: Opioid efficacy, bias, and selectivity

Drugs acting at the opioid receptor family are clinically used to treat chronic and acute pain, though they represent the second line of treatment behind GABA analogs, antidepressants and SSRI\u27s. Within the opioid family mu and kappa opioid receptor are commonly targeted. However, activation of the mu opioid receptor has side effects of constipation, tolerance, dependence, euphoria, and respiratory depression; activation of the kappa opioid receptor leads to dysphoria and sedation. The side effects of mu opioid receptor activation have led to mu receptor drugs being widely abused with great overdose risk. For these reasons, newer safer opioid analgesics are in high demand. For many years a focus within the opioid field was finding drugs that activated the G protein pathway at mu opioid receptor, without activating the β-arrestin pathway, known as biased agonism. Recent advances have shown that this may not be the way forward to develop safer analgesics at mu opioid receptor, though there is still some promise at the kappa opioid receptor. Here we discuss recent novel approaches to develop safer opioid drugs including efficacy vs bias and fine-tuning receptor activation by targeting sub-pockets in the orthosteric site, we explore recent works on the structural basis of bias, and we put forward the suggestion that Gα subtype selectivity may be an exciting new area of interest

Efforts towards the synthesis of furan containing bioactive compounds

Furan is a valuable subunit in pharmaceutical chemistry. However, there are still challenges in synthesizing furan-containing compounds. Two approaches have been attempted to address this issue. 1. Using enynal molecules as a carbene precursor for synthesizing functionalized furyl-pyrrolidines. A cascade approach was developed for synthesizing functionalized (2-furyl)-2-pyrrolidines, showcasing both convergence and remarkable stereoselectivity. This domino process proceeds through an N–H insertion into enynal-derived metal-carbenoid, followed by an intramolecular aldol reaction to provide pyrrolidines with high diastereoselectivity (>98:2). This chemistry utilizes Earthabundant zinc chloride as a catalyst with loading as low as 1 mol%. This method operates under mild conditions and demonstrates high chemoselectivity by accommodating substrates bearing functionalities such as free alcohols, alkenes, and alkynes. 2 Towards the total synthesis of collybolide. Collybolide is a natural product that was first isolated from the fungus Collybia maculata. It has attracted attention due to its potential therapeutic applications, particularly in the treatment of pain and inflammation. Its complex structure, however, makes it a challenging target for total synthesis. Our route starts from simple glutamic acid. This route aimed to minimize the use of chiral reagents and catalysts to install all 6 stereocenters in Collybolide. So far, after 11 reactions, we have achieved the intermediate having 15 out of 22 carbon atoms and 4 out of 6 stereocenters in Collybolide without using any chiral reagents and catalysts other than glutamic acid. One of the significances of this route is that different from the traditional synthetic route, we installed furan moiety at a very early stage. Furan is known for its instability, however, in our route, it is stable throughout the synthetic pathway

Docking 14 Million Virtual Isoquinuclidines against the μ and κ Opioid Receptors Reveals Dual Antagonists–Inverse Agonists with Reduced Withdrawal Effects

Large library docking of tangible molecules has revealed potent ligands across many targets. While make-on-demand libraries now exceed 75 billion enumerated molecules, their synthetic routes are dominated by a few reaction types, reducing diversity and inevitably leaving many interesting bioactive-like chemotypes unexplored. Here, we investigate the large-scale enumeration and targeted docking of isoquinuclidines. These “natural-product-like” molecules are rare in current libraries and are functionally congested, making them interesting as receptor probes. Using a modular, four-component reaction scheme, we built and docked a virtual library of over 14.6 million isoquinuclidines against both the μ- and κ-opioid receptors (MOR and KOR, respectively). Synthesis and experimental testing of 18 prioritized compounds found nine ligands with low μM affinities. Structure-based optimization revealed low- and sub-nM antagonists and inverse agonists targeting both receptors. Cryo-electron microscopy structures illuminate the origins of activity on each target. In mouse behavioral studies, a potent joint MOR-antagonist and KOR-inverse-agonist reversed morphine-induced analgesia, phenocopying the MOR-selective antioverdose agent naloxone. Encouragingly, the isoquinuclidine induced less severe opioid-withdrawal symptoms versus naloxone and did not induce conditioned-place aversion, reflecting reduced dysphoria, consistent with its KOR-inverse agonism. The strengths and weaknesses of bespoke library docking and of docking for opioid receptor polypharmacology will be considered

Pre-clinical Anti-Addictive and Side-Effect profiles of Novel Kappa-opioid Agonists

Background: Drug addiction is a chronic, relapsing disorder with great socioeconomic and morbidity costs. An estimated 27 million people worldwide suffer from drug dependence, with over 180,000 drug abuse-related deaths reported annually (UNODC, 2015). Currently, there are no FDA-approved pharmacotherapies for psychostimulant addiction, limiting the efficacy of treatment for cocaine and amphetamine abuse. Kappa-opioid receptor (KOPr) agonists can act as inhibitors of reward, and have been investigated in pre-clinical models of drug abuse for potential anti-addictive properties, but display undesirable side-effects such as dysphoria and sedation. A naturally-occurring KOPr agonist, Salvinorin A (SalA), has been explored as a lead for new KOPr-based anti-addictive medications. SalA is a short-acting but potent non-nitrogenous KOPr agonist with known anti-cocaine effects, and chemical alterations to this structure have produced novel agonists with comparable or greater potency at the KOPr. This thesis compares two novel SalA analogues, 16-ethynyl Salvinorin A (Ethy-SalA) and 16-methyl Salvinorin A (Me-SalA), in pre-clinical models of addiction and side-effect tests. Methods: Sprague-Dawley rats were used to model the behavioural effects of acute KOPr treatment upon cocaine self-administration and drug-seeking behaviour, natural reward-seeking, cocaine-induced and spontaneous locomotion, and pro-depressive forced-swim testing. Transiently co-transfected HEK-293 cells were used to model the influence of KOPr activation upon dopamine transporter (DAT) function in an in vitro model of dopamine uptake, using confocal microscopy to detect internalisation of the fluorescent DAT substrate ASP+. Results: Acute pre-treatments of Ethy-SalA significantly attenuated cocaine-reinstatement of drug-seeking behaviour (at 0.1 and 0.3 mg/kg) and progressive ratio (PR) self-administration of cocaine (at 2.0 mg/kg). The less potent agonist Me-SalA did not attenuate cocaine-reinstatement or PR self-administration at the doses tested (0.3-2.0 mg/kg). Despite apparent anti-cocaine effects, Ethy-SalA (0.3 mg/kg) was not found to effectively reduce cocaine-induced locomotor hyperactivity or sensitisation in rats. Side-effect screens were carried out on the novel compounds using the doses tested in cocaine-primed reinstatement. Ethy-SalA (0.3 mg/kg) and Me-SalA (1.0 mg/kg) did not significantly affect spontaneous locomotor behaviour 0.3 mg/kg, or reduce self-administration of the natural reward sucrose at a dose of 0.3 mg/kg in rats. Depression-like effects caused by acute Ethy-SalA treatment (0.3 mg/kg) were also not detected in the Forced Swim Test. Treatment with Ethy-SalA (10 µM) significantly increased uptake of the fluorescent ASP+ in co-transfected DAT/KOPr HEK-293 cells. Conclusions: A single treatment of the novel KOPr agonist Ethy-SalA, but not the novel agonist Me-SalA, was found to attenuate drug-seeking behaviours in models of cocaine administration with greater potency than SalA, and without detectable sedative or depression-like effects at a dose of 0.3 mg/kg. The cellular mechanism-of-action by which Ethy-SalA depresses cocaine reward is at least in part due to positive regulation of DAT, which would act to reduce extracellular dopamine within the brain. The lack of significant side-effects and the apparent improved potency of the compound support further exploration of Ethy-SalA as a lead for the development of an anti-addictive pharmacotherapy

New senthesis and derivatizations of peptidomimetics and peptide-conjugates for theranostic applications

In the contest of a modern green chemistry approach, we firstly tried to substituent the classic peptide synthesis approach with the use of N-carboxyanhydrides in the presence of Hydroxyapatite, a high biocompatible inorganic base. Despite the great results, further developments are necessary for a daily use in laboratory and for our research, we decided to proceed with solid phase or liquid phase synthesis. In the first chapter, the treatment of pain with the use of opioids is introduced. The abuse and misuse of these kind of potent analgesics, led to the necessity of developing new drugs with less side effects. Starting from a previous study, where the introduction of a lactam-like structure in the place of the proline of Endomorphine1, switched the selectivity from MOR to KOR, we designed and synthetized three different libraries by placing a different trans inducer element to gain the desired selectivity and activity forcing the structure to adopt a linear rather than folded position. In the second chapter, we focused on lactate dehydrogenase, an enzyme overexpressed when the cells in hypoxia conditions, like in a tumour mass, need to produce energy through the transformation of pyruvate into lactate. We synthetized different cyclic peptidomimetics, designed to be inhibitors, as powerful tool to contrast cancer cells growing. Biological assays produced satisfactory preliminary results, but further studies are necessary for a definitive output. Finally in the last chapter, the cancer treatment problem is also approached through the design of nanoparticles, able to deliver drugs with efficacy and selectivity. We firstly synthetized silica core nanoparticles, built with toxic peptide sequences conjugated through click chemistry with Pluronic acid and then, in collaboration with Miriam Royo’s research group, we synthetized multivalent platforms for used drugs for the treatment of advanced colorectal cancer

Design and structural validation of peptide-drug conjugate ligands of the kappa-opioid receptor

Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-β-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-β-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-β-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions

Design and synthesis of cyclic analogs of the kappa opioid receptor antagonist arodyn

Opioid receptors are important therapeutic targets for mood disorders and pain. Kappa opioid receptor (KOR) antagonists have recently shown potential for treating drug addiction and depression. Arodyn (Ac[Phe1,2,3,Arg4,D-Ala8]Dyn A(1-11)-NH2), an acetylated dynorphin A (Dyn A) analog, has demonstrated potent and selective KOR antagonism, but can be rapidly metabolized by proteases. Cyclization of arodyn could enhance metabolic stability and potentially stabilize the bioactive conformation to give potent and selective analogs. Accordingly, novel cyclization strategies utilizing ring closing metathesis (RCM) were pursued. However, side reactions involving olefin isomerization of O-allyl groups limited the scope of the RCM reactions, and their use to explore structure-activity relationships of aromatic residues. Here we developed synthetic methodology in a model dipeptide study to facilitate RCM involving Tyr(All) residues. Optimized conditions that included microwave heating and the use of isomerization suppressants were applied to the synthesis of cyclic arodyn analogs. Initial pharmacological data indicates the constraints involving aromatic residues were generally well tolerated at KOR with most of the analogs exhibiting affinities within 3- to 4-fold that of arodyn. RCM was also used in the synthesis of head to side chain cyclized arodyn analogs. Attempted cyclizations involving Tyr(All) residues proceeded in low yields, in contrast to cyclizations involving AllGly residues. However, ring contraction products as a result of olefin isomerization were also observed during the latter cyclizations. The resulting head to side chain cyclized arodyn analogs exhibited a 5-fold decrease in KOR affinity compared to arodyn. We further explored synthesis of arodyn analogs cyclized in both the N-terminal and C-terminal segments resulting in bicyclic arodyn analogs. Here, we present the synthesis of two initial bicyclic peptide KOR ligands with different topologies. The RCM-based bicyclic arodyn analog exhibited KOR affinity within 3-fold that of arodyn, whereas the lactam-based bicyclic analog displayed a substantial loss in affinity for KOR. There are currently no reports of bicyclic opioid peptide ligands and such bicyclic arodyn analogs could be useful as pharmacological tools