Bivalent Ligands Research Articles

The bivalent ligand, MMG22, reduces neuropathic pain after nerve injury without the side effects of traditional opioids.

Functional interactions between the mu opioid receptor (MOR) and the metabotropic glutamate receptor 5 (mGluR5) in pain and analgesia have been well established. MMG22 is a bivalent ligand containing MOR agonist (oxymorphamine) and mGluR5 antagonist (MPEP) pharmacophores tethered by a 22-atom linker. MMG22 has been shown to produce potent analgesia in several models of chronic inflammatory and neuropathic pain (NP). This study assessed the efficacy of systemic administration of MMG22 at reducing pain behavior in the spared nerve injury (SNI) model of NP in mice, as well as its side-effect profile and abuse potential. MMG22 reduced mechanical hyperalgesia and spontaneous ongoing pain after SNI, with greater potency early (10 days) as compared to late (30 days) after injury. Systemic administration of MMG22 did not induce place preference in naive animals, suggesting absence of abuse liability when compared to traditional opioids. MMG22 also lacked the central locomotor, respiratory, and anxiolytic side effects of its monomeric pharmacophores. Evaluation of mRNA expression showed the transcripts for both receptors were colocalized in cells in the dorsal horn of the lumbar spinal cord and dorsal root ganglia. Thus, MMG22 reduces hyperalgesia after injury in the SNI model of NP without the typical centrally mediated side effects associated with traditional opioids.

Kisspeptin Amplifies GLP‐1 Potentiation of Insulin Secretion by a cAMP Independent Pathway

Glucagon Like Peptide‐1 (GLP‐1) and Kisspeptin (KP) receptors are co‐expressed on pancreatic b‐cells, and activation of both receptors potentiates glucose stimulated insulin secretion (GSIS). Unlike GLP‐1, KP has a low affinity for receptor activation (EC50 GLP‐1 ~20 nM vs KP >1 uM). The effects of GLP‐1 and a 10 amino acid analog of KP (KP10) on insulin secretion and cell signaling were studied in the INS 832/3 cell line. By itself KP10 has no effect on insulin secretion in the absence of glucose, but doubled GSIS (GSIS = 37.7 +/− 6.7; +KP10 = 69.2 +/− 1.9 ng/ml/80min) with an EC50 of ~5–10 mM. At saturating doses, KP10 (100 mm) and GLP‐1 (100 nM) had similar effects on GSIS, and in combination had an additive effect (GLP‐1 = 49.0 +/− 2.0, GLP‐1 + KP10 = 63.5 +/− 5.6 ng/ml/80min) consistent with activation of independent downstream signaling pathways. Neither GLP‐1 or KP‐10 altered intracellular Ca2+ levels at basal or activating glucose concentrations. GLP‐1 increased cAMP production (165.0 +/− 17.4 % over control), whereas KP‐10 was without effect. Conversely, pretreatment with KP‐10 enhanced glucose stimulated oxygen consumption and mitochondrial NADH fluorescence, although both responses were inconsistent. Therefore, the effects of KP on b‐cell metabolic activity needs to be investigated in more detail.We also developed a bivalent ligand composed of GLP‐17‐36 and KP10 binding domains, linked with a 3‐mer of proline/glycine repeats, to evaluate if linking KP10 to a high affinity ligand could promote KP10 activity at low concentrations by presumably increasing its local membrane concentration after GLP‐1 binding. The dimer also did not activate secretion in the absence of glucose, but doubled GSIS much like the individual monomers with an EC50 ~200nM; that is, an additive response was not obvious. Currently, we are determining the ability of GLP‐1/KP10 to initiate downstream signaling from both ligands to resolve the question: is KP active in the GLP‐1/KP at concentrations where monomeric KP10 is not.Support or Funding InformationFunded by the American Diabetes Association and Juvenile Diabetes Research Foundation

A Novel Bivalent Mannosylated Targeting Ligand Displayed on Nanoparticles Selectively Targets Anti-Inflammatory M2 Macrophages.

Persistent activation of macrophages (MP)s into a proinflammatory M1 or anti-inflammatory M2 phenotype plays a role in several pathological conditions, including autoimmune diseases, fibrosis, infections, atherosclerosis and tumor development. The mannose receptor (MR, CD206), expressed at low levels on resting MPs and absent on M1 MPs, is highly expressed on M2 MPs, making it a potential target and drug delivery portal. Recently, we developed a novel, highly selective MR targeting ligand (MRTL), consisting of two mannose molecules separated by a monodisperse 12 unit poly(ethylene glycol) linker, to enhance the cellular uptake of polymeric nanocarriers. The feasibility of using the MRTL ligand for selectively targeting M2 MPs for intracellular delivery of nanoparticles (NPs) was investigated. Rat peritoneal MPs were differentiated into an M1 or M2 phenotype using IFN-γ and IL-4/IL-13, respectively. Expression of the M1 marker, inducible nitric oxide synthase (iNOS), and the M2 markers arginase (Arg)-1 and MR (at both the mRNA and protein levels) confirmed MP phenotypic activation. Resting, M1 and M2 MPs were treated with fluorescein isothiocyanate (FITC)-labeled MRTL or NPs displaying FITC-labeled MRTL at two surface densities (1 and 10%) and examined by confocal microscopy. Intracellular fluorescence was also quantified. Uptake of the MRTL was 2.4- and 11.8-fold higher in M2 MPs when compared to resting or M1 MPs, respectively, consistent with marker expression levels. Mannan, a competitive inhibitor of the MR, abrogated MRTL uptake. MRTL also co-localized with a fluid-phase endocytosis marker, further suggesting that uptake was mediated by MR-mediated endocytosis. Intracellular NP fluorescence was confirmed by flow cytometry and by confocal microscopy. MRTL-NPs accumulated intracellularly with no significant cell surface binding, suggesting efficient translocation. NPs displaying a low surface density (1%) of the MRTL exhibited significantly higher (2.3-fold) uptake into M2 MPs, relative to resting and M1 MPs. The 10% MRTL-NPs displayed greater uptake by M2 MPs when compared to resting and M1 MPs, but less uptake than 1% MRTL-NPs into M2 MPs. Control FITC-labeled plain NPs did not exhibit selective MP uptake. These studies demonstrate that M2 MPs are selectively targeted by NPs displaying a novel bivalent ligand that utilizes the MR as a target/portal for cell entry. This study also establishes the feasibility of the approach allowing for further investigation in vivo.

Design and synthesis of a bivalent probe targeting the putative mu opioid receptor and chemokine receptor CXCR4 heterodimer.

Opioid abuse and HIV/AIDS have been defined as synergistic epidemics. Opioids can accelerate HIV replication in the immune system by up-regulating the expression of HIV co-receptor CXCR4. Several hypotheses have been suggested as the mechanism of CXCR4 modulation by opioids through their activation on the mu opioid receptor (MOR). One hypothesis is the putative heterodimerization of the MOR and CXCR4 as a mechanism of cross-talk and subsequent exacerbation of HIV replication. Bivalent chemical probes can be powerful molecular tools to characterize protein-protein interactions, and modulate the function related to such interactions. Herein we report the design and synthesis of a novel bivalent probe to explore the putative MOR-CXCR4 dimerization and its potential pharmacological role in enhancing HIV progression. The developed bivalent probe was designed with two distinct pharmacophores linked through a spacer. One pharmacophore (naltrexone) will interact with the MOR and the other (IT1t) with the CXCR4. The overall synthetic routes to prepare the bivalent probe and its corresponding monovalent controls were comprised of 18-22 steps with acceptable yields. Preliminary biological evaluation showed that the bivalent probe preserved binding affinity and functional activity at both respective receptors, supporting the initial molecular design.

Differential allosteric modulation within dopamine D2R - neurotensin NTS1R and D2R - serotonin 5-HT2AR receptor complexes gives bias to intracellular calcium signalling

Proceeding investigations of G protein-coupled receptor (GPCR) heterocomplexes have demonstrated that the dopamine D2 receptor (D2R), one of the hub receptors in the physiology of schizophrenia, interacts with both the neurotensin NTS1 (NTS1R) and the serotonin 5-HT2A receptor (5-HT2AR) in cell lines and rodent brain tissue. In situ proximity ligation assay and BRET-based saturation experiments confirmed interacting receptor assemblies in HEK293T and neuronal HT22 cells. The NTS1R agonist NT(8-13) reduces the Gαq-mediated calcium signal in the NTS1R-D2R complex compared to the NTS1R monomer which could be reversed by D2R antagonists. The bivalent ligand CS148 (NTS1R-agonistic, D2R-antagonistic) increased the calcium response addressing the dimer, consistent with the effect of the monovalent ligands suggesting an allosteric D2R-mediated modulation. In contrast, the 5-HT2AR-D2R heteromer did not show a calcium-altering receptor-receptor interaction. Despite their common coupling-preference for Gαq, 5-HT2AR and NTS1R supposedly interact with D2R each in a unique mode. This remarkably diverse ligand-mediated signalling in two different D2R heteroreceptor complexes illustrates the complexity of receptor-receptor interactions and their potential of modifying cell responses to external stimuli. Therefore, GPCR heteromers may provide a very promising novel target for the therapy of neuropsychiatric disorders.

Binding of Monovalent and Bivalent Ligands by Transthyretin Causes Different Short- and Long-Distance Conformational Changes.

The wild type protein, transthyretin (TTR), and over 120 genetic TTR variants are amyloidogenic and cause, respectively, sporadic and hereditary systemic TTR amyloidosis. The homotetrameric TTR contains two identical thyroxine binding pockets, occupation of which by specific ligands can inhibit TTR amyloidogenesis in vitro. Ligand binding stabilizes the tetramer, inhibiting its proteolytic cleavage and its dissociation. Here, we show with solution-state NMR that ligand binding induces long-distance conformational changes in the TTR that have not previously been detected by X-ray crystallography, consistently with the inhibition of the cleavage of the DE loop. The NMR findings, coupled with surface plasmon resonance measurements, have identified dynamic exchange processes underlying the negative cooperativity of binding of “monovalent” ligand tafamidis. In contrast, mds84, our prototypic “bivalent” ligand, which is a more potent stabilizer of TTR in vitro that occupies both thyroxine pockets and the intramolecular channel between them, has greater structural effects.

A solid-phase method for synthesis of dimeric and trimeric ligands: Identification of potent bivalent ligands of 14-3-3σ

Multivalent protein-protein interactions including bivalent and trivalent interactions play a critical role in mediating a wide range of biological processes. Hence, there is a significant interest in developing molecules that can modulate those signaling pathways mediated by multivalent interactions. For example, multimeric molecules capable of binding to a receptor protein through a multivalent interaction could serve as modulators of such interactions. However, it is challenging to efficiently generate such multimeric ligands. Here, we have developed a facile solid-phase method that allows for the rapid generation of (homo- and hetero-) dimeric and trimeric protein ligands. The feasibility of this strategy was demonstrated by efficiently synthesizing fluorescently-labeled dimeric peptide ligands, which led to dramatically increased binding affinities (~400-fold improvement) relative to a monomeric 14-3-3σ protein ligand.

Targeting MOR-mGluR5 heteromers reduces bone cancer pain by activating MOR and inhibiting mGluR5

Pain is among the most common symptoms in cancer and approximately 90% of patients experience end-stage cancer pain. The management of cancer pain is challenging due to the significant side effects associated with opioids, and novel therapeutic approaches are needed. MMG22 is a bivalent ligand containing MOR agonist and mGluR5 antagonist pharmacophores joined by a 22-atom spacer. MMG22 exhibited extraordinary analgesia following intrathecal administration in a mouse model of bone cancer pain. Here, we assessed the effectiveness of systemic administration of MMG22 in reducing cancer pain and evaluated whether MMG22 displays side effects associated with opioids. Fibrosarcoma cells were injected into and around the calcaneus bone in C3H mice. Mechanical hyperalgesia was defined as an increase in the paw withdrawal frequencies (PWFs) evoked by application of a von Frey monofilament (3.9 mN bending force) applied to the plantar surface of the hind pawSubcutaneous (s.c.), intramuscular (i.m.), and oral (p.o.) administration of MMG22 produced robust dose-dependent antihyperalgesia, whose ED50 was orders of magnitude lower than morphine. Moreover, the ED50 for MMG22 decreased with disease progression. Importantly, s.c. administration of MMG22 did not produce acute (24 h) or long-term (9 days) tolerance, was not rewarding (conditioned place preference test), and did not produce naloxone-induced precipitated withdrawal or alter motor function. A possible mechanism of action of MMG22 is discussed in terms of inhibition of spinal NMDAR via antagonism of its co-receptor, mGluR5, and concomitant activation of neuronal MOR. We suggest that MMG22 may be a powerful alternative to traditional opioids for managing cancer pain.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Abstract 1294: Differential sigma-2 receptor-mediated metabolic stimulative and apoptotic effects of monovalent CM571 and its homo-bivalent counterpart, MAM03055A

Abstract Sigma-2 receptors, recently identified as TMEM97, are highly expressed in various types of cancer cells. Sigma-2 receptor agonists have traditionally been characterized as ligands that induce programmed cell death in various cancer cell types. Sigma-2 receptor-mediated cell death involves a number of mechanisms including caspase activation, mitochondrial depolarization, and autophagy. Recently, we reported a novel metabolically stimulative function of the sigma-2 receptor with stimulation of glycolytic hallmarks, including increase in cellular ATP level, reduced ROS, and stabilization of HIF-1α. We have investigated a family of 6-substituted analogs of the canonical sigma-2 receptor antagonist, SN79, members of which can produce both the metabolic stimulative and the cytotoxic effects. Here we compare the activities of two closely related compounds. CM571, the 6-amino derivative, bound with high affinity to both sigma-1 and sigma-2 receptors (sigma-1 Ki = 15.5 nM; sigma-2 Ki = 21.7 nM). MAM03055A (MAM) is essentially a homo-bivalent dimer of CM571, linked by a thiourea moiety. MAM exhibited high affinity and selectivity for sigma-2 receptors (sigma-1 Ki = 3,371 nM; sigma-2 Ki = 55.9 nM). The functional effects of the two compounds were compared in human SK-N-SH neuroblastoma cells. CM571 caused a dose-dependent stimulation of MTT reduction, indicating the metabolic stimulative-effect, producing 40% stimulation at 30 µM. By contrast, MAM induced dose-dependent cell death with an EC50 = 4.24 µM. MAM induced time-dependent cleavage of proapoptotic Bid, a key component of the programmed cell death mechanism of sigma-2 ligands in these cells. Previous studies had shown that only irreversibly binding, isothiocyanate analogs of SN79 induced programmed cell death, while other analogs produced metabolic stimulation. Rat liver membranes were treated with compound, washed to remove unbound ligand, and then subjected to radioligand binding to determine recovery of sigma-1 receptors ([3H](+)-pentazocine) and sigma-2 receptors ([3H]DTG + (+)-pentazocine). Treatment with CM571 resulted in full recovery of both sigma-1 and sigma-2 binding. However, MAM treatment resulted in loss of sigma-2 binding and full recovery of sigma-1 activity, indicating irreversible binding to the sigma-2 receptor. Since there is no chemically reactive moiety in MAM to react covalently, MAM binds “pseudo-irreversibly”. Treatment of cells with MAM for 60 min, followed by washout produced nearly comparable levels of cell death 24 h later as continuous exposure of cells for 24 hours, consistent with pseudo-irreversible action at the sigma-2 receptor. The results show that mono-valent and bivalent sigma-2 ligands in this series show different modes of interaction with the receptor, resulting in divergent effects on cell viability. Citation Format: Cheri Z. Liu, Bridget M. McVeigh, Marco Mottinelli, Hilary E. Nicholson, Christopher R. McCurdy, Wayne D. Bowen. Differential sigma-2 receptor-mediated metabolic stimulative and apoptotic effects of monovalent CM571 and its homo-bivalent counterpart, MAM03055A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1294.

Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling.

Deregulation of innate immune TLR4 signaling contributes to various diseases including neuropathic pain and drug addiction. Naltrexone is one of the rare TLR4 antagonists with good blood-brain barrier permeability and showing no stereoselectivity for TLR4. By linking 2 naltrexone units through a rigid pyrrole spacer, the bivalent ligand norbinaltorphimine was formed. Interestingly, (+)-norbinaltorphimine [(+)-1] showed ∼25 times better TLR4 antagonist activity than naltrexone in microglial BV-2 cell line, whereas (-)-norbinaltorphimine [(-)-1] lost TLR4 activity. The enantioselectivity of norbinaltorphimine was further confirmed in primary microglia, astrocytes, and macrophages. The activities of meso isomer of norbinaltorphimine and the molecular dynamic simulation results demonstrate that the stereochemistry of (+)-1 is derived from the (+)-naltrexone pharmacophore. Moreover, (+)-1 significantly increased and prolonged morphine analgesia in vivo. The efficacy of (+)-1 is long lasting. This is the first report showing enantioselective modulation of the innate immune TLR signaling.-Zhang, X., Peng, Y., Grace, P. M., Metcalf, M. D., Kwilasz, A. J., Wang, Y., Zhang, T., Wu, S., Selfridge, B. R., Portoghese, P. S., Rice, K. C., Watkins, L. R., Hutchinson, M. R., Wang, X. Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling.

Potent Acetylcholinesterase Selective and Reversible Homodimeric Agent Based on Tacrine for Theranostics.

Acetylcholinesterase (AChE) has been an important biomarker for diagnosing Alzheimer's disease (AD), due to reduction in AChE activity in post-mortem brains of AD patients. A potent, selective, and reversible homodimeric inhibitor of AChE, 5-amino- N1, N3-bis(2-(1,2,3,4-tetrahydroacridin-9-ylamino)ethyl)isophthalamide (compound 4), was synthesized by using 9-alkyl(1,2,3,4-tetrahydroacridine) pharmacophore with appended functionality. In the present work, we report the synthesis of this bivalent inhibitor of AChE. The homodimeric ligand structure was designed and studied with molecular docking tools, which revealed its high affinity and interactions with active site gorge of AChE, which includes both catalytic active site (CAS) and peripheral active site (PAS). The IC50 value of this bivalent inhibitor for AChE and BuChE were 0.54 ± 0.06 and 32.49 ± 1.2 nM, respectively, with a selectivity ratio of 60.16 toward AChE. The designed ligand also showed potent inhibitory properties on PAS activity as well as on AChE-induced amyloid aggregation with low cytotoxicity on rat hippocampal neurons. The AFM images further corroborated the Aβ1-42 aggregation inhibition by compound 4 to an extent similar to bis(7)-tacrine. Moreover, the bivalent ligand was also proven to be of neurogenic potential due to its ability to induce S-phase post-treatment in rat hippocampal neuronal cells. On the basis of initial results, the agent could be further explored for its theranostic value clinically, which gives the possibility of tracing the AChE levels by molecular imaging techniques in correlation with progression of neurocognitive disorders like AD for better therapy response and patient management.

The bivalent ligand MCC22 potently attenuates hyperalgesia in a mouse model of cisplatin-evoked neuropathic pain without tolerance or reward

Cisplatin and other widely employed platinum-based anticancer agents produce chemotherapy-induced peripheral neuropathy (CIPN) that often results in pain and hyperalgesia that are difficult to manage. We investigated the efficacy of a novel bivalent ligand, MCC22, for the treatment of pain arising from CIPN. MCC22 consists of mu opioid receptor (MOR) agonist and chemokine receptor 5 (CCR5) antagonist pharmacophores connected through a 22-atom spacer and was designed to target a putative MOR-CCR5 heteromer localized in pain processing areas. Mice received once daily intraperitoneal (i.p.) injections of cisplatin (1 mg/kg) for seven days and behavior testing began 7 days later. Cisplatin produced mechanical hyperalgesia that was decreased dose-dependently by MCC22 given by intrathecal (ED50 = 0.004 pmol) or i.p. (3.07 mg/kg) routes. The decrease in hyperalgesia was associated with decreased inflammatory response by microglia in the spinal cord. Unlike morphine, MCC22 given daily for nine days did not exhibit tolerance to its analgesic effect and its characteristic antihyperalgesic activity was fully retained in morphine-tolerant mice. Furthermore, MCC22 did not alter motor function and did not exhibit rewarding properties. Given the exceptional potency of MCC22 without tolerance or reward, MCC22 has the potential to vastly improve management of chronic pain due to CIPN.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Novel sumanirole bivalent analogues as potent dopamine D2 receptor Go‐protein biased agonists

The dopamine D2 receptor (D2R) is a major target for therapeutic development of treatments for neuropsychiatric disorders. In particular, D2R agonists have been used for neurological disorders such as restless leg syndrome and Parkinson's Disease. However, current D2R agonist pharmacotherapies have drawbacks including the development of tolerance and other adverse side‐effects. To potentially improve D2R agonist‐based medications, we have designed bivalent ligands that have two pharmacophores tethered by a link. This strategy aims to improve selectivity, affinity, and cell signaling activation bias. Indeed, it has been demonstrated that the secondary pharmacophore plays a key role in inducing activation bias towards either G protein, or β‐arrestin pathways.Here we characterized a series of bivalent orthosteric‐arthosteric heteroaryl pharmacophores with assorted linkers and compared them with the potent orthosteric D2R‐selective agonist, sumanirole. Several bivalent analogues demonstrated modest increases in D2R affinity compared to sumanirole. D2R functional assays using bioluminescence resonance energy transfer (BRET) showed improved efficacy and potency for G protein coupling, but substantially diminished efficacy and potency for β‐arrestin recruitment. Analysis using an operational model of bias revealed significant G‐protein activation bias (>1000‐fold). Further, several compounds exhibited a G protein subtype (GoA over Gi1) activation bias. Finally, D2R‐mediated, G protein‐coupled inwardly‐rectifying potassium channel (GIRK) activity was measured in midbrain slices using electrophysiology. Recordings from D2R‐expressing dopaminergic cells in the ventral tegmental area confirmed prolonged GIRK activation by the biased bivalent compounds, compared to dopamine or sumanirole.Our novel sumanirole bivalent analogues demonstrate a proof of concept for G protein activation bias and thus may lead to D2R agonist therapeutics with reduced tolerance and improved efficacy. With these novel tools, the preferential targeting and activation of Go‐proteins, the most predominant Gi‐like protein family in the brain, are now conceivable in animal models. This advance may aid in treatments of basal ganglia disorders with reductions in untoward side‐effects in humans.Support or Funding InformationWork was supported by the Intramural funds of the National Institute on Drug AbuseThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The TwistDock workflow for evaluation of bivalent Smac mimetics targeting XIAP.

Purpose: Mimetics based on Smac, the native inhibitor of XIAP, are promising drug-candidates for the treatment of cancer. Bivalent Smac mimetics inhibit XIAP with even higher potency than monovalent mimetics, but how to optimize the linker that tethers the two monovalent binding motifs remains controversial.Methods: To construct an ensemble of bivalent complex structures for evaluating various linkers, we propose herein a workflow, named TwistDock, consisting of steps of monovalent docking and linker twisting, in which the degrees of freedom are sampled focusing on the rotation of single bonds of the linker.Results: The obtained conformations of bivalent complex distribute randomly in the conformational space with respect to two reaction coordinates introduced by the linker, which are the distance of the two binding motifs and the dihedral angle of the two planes through the linker and each of the binding motifs. Molecular dynamics starting from 10 conformations with the lowest enthalpy of every complex shows that the conformational tendency of the complex participated by compound 9, one of the compounds with the largest binding affinity, is distinct from others. By umbrella sampling of the complex, we find its global minimum of the free energy landscape. The structure shows that the linker favors a compact conformation, and the two BIR domains of XIAP encompass the ligand on the opposite sides.Conclusion: TwistDock can be used in fine-tuning of bivalent ligands targeting XIAP and similar receptors dimerized or oligomerized.

Development of the 99mTc-Hydroxamamide Complex as a Probe Targeting Carbonic Anhydrase IX.

Carbonic anhydrase IX (CA-IX) is regarded as a favorable target for in vivo imaging because of its specific expression in hypoxic regions of tumors. Hypoxia assists tumor propagation and growth and is resistant to chemotherapy and radiotherapy. Here, we designed and synthesized [99mTc]hydroxamamide ([99mTc]Ham) and [99mTc]methyl-substituted-hydroxamamide ([99mTc]MHam) complexes including a bivalent CA-IX ligand, sulfonamide (SA), and ureidosulfonamide (UR). In a cell binding assay, [99mTc]Ham complexes with bivalent SA ([99mTc]SAB2A and [99mTc]SAB2B) and UR ([99mTc]URB2A and [99mTc]URB2B) showed significantly greater uptake into CA-IX high-expressing (HT-29) cells than that into CA-IX low-expressing cells. Since the binding affinity of [99mTc]URB2A and [99mTc]URB2B for CA-IX was significantly higher than that of [99mTc]SAB2A and [99mTc]SAB2B, we additionally synthesized [99mTc]MURB2 (a [99mTc]MHam complex with bivalent UR) and evaluated the CA-IX-specific binding affinity of [99mTc]URB2A, [99mTc]URB2B, and [99mTc]MURB2. Their uptake into HT-29 cells was reduced by the addition of a CA inhibitor, acetazolamide, suggesting their CA-IX-specific binding affinity. A biodistribution study in HT-29 tumor-bearing mice was carried out using [99mTc]URB2A and [99mTc]MURB2 with the highest specificity for HT-29 cells. [99mTc]URB2A showed moderate tumor uptake and reduction by coinjection with acetazolamide; however, the tumor/blood ratio was insufficient for in vivo imaging. These results provided key information for the design of novel Ham-based imaging probes targeting CA-IX.

O-linked melatonin dimers as bivalent ligands targeting dimeric melatonin receptors

A series of dimeric melatonin analogues 3a-e obtained by connecting two melatonin molecules through the methoxy oxygen atoms with spacers spanning 16–24 atoms and the agomelatine dimer 7 were synthesized and characterized in 2-[125-I]-iodomelatonin binding assays, bioluminescence resonance energy transfer (BRET) experiments, and in functional cAMP and β-arrestin recruitment assays at MT1 and MT2 receptors. The binding affinity of 3a-e generally increased with increasing linker length. Bivalent ligands 3a-e increased BRET signals of MT1 dimers up to 3-fold compared to the monomeric control ligand indicating the simultaneous binding of the two pharmacophores to dimeric receptors. Bivalent ligands 3c and 7 exhibited important changes in functional properties on the Gi/cAMP pathway but not on the β-arrestin pathway compared to their monomeric counterparts. Interestingly, 3c (20 atoms spacer) shows inverse agonistic properties at MT2 on the Gi/cAMP pathway. In conclusion, these findings indicate that O-linked melatonin dimers are promising tools to develop signaling pathway-based bivalent melatonin receptor ligands.

Emerging Insights into Mu Opioid Pharmacology.

Opioid analgesics, most of which act through mu opioid receptors, have long represented valuable therapeutic agents to treat severe pain. Concerted drug development efforts for over a 100years have resulted in a large variety of opioid analgesics used in the clinic, but all of them continue to exhibit the side effects, especially respiratory depression, that have long plagued the use of morphine. The recent explosion in fatalities resulting from overdose of prescription and synthetic opioids has dramatically increased the need for safer analgesics, but recent developments in mu receptor research have provided new strategies to develop such drugs. This chapter reviews recent advances in developing novel opioid analgesics from an understanding of mu receptor structure and function. This includes a summary of the mechanism of agonist binding deduced from the crystal structure of mu receptors. It will also highlight the development of novel agonist mechanisms, including biased agonists, bivalent ligands, and allosteric modulators of mu receptor function, and describe how receptor phosphorylation modulates these pathways. Finally, it will summarize research on the alternative pre-mRNA splicing mechanisms that produces a multiplicity of mu receptor isoforms. Many of these isoforms exhibit different pharmacological specificities and brain circuitry localization, thus providing an opportunity to develop novel drugs with increased therapeutic windows.

Synthetic route towards potential bivalent ligands possessing opioid and D2/D3 pharmacophores

A scalable, cost-efficient and simple synthetic pathway towards potential bivalent opioid/dopamine receptor ligands was developed and optimized. Three novel compounds that contain both opioid and dopamine pharmacophores linked by the four methylene group chain were synthesized in 33, 35 and 39 % overall yield after a four-step synthetic route starting from three commercially available N-aryl piperazines. The anilino piperidine precursor was easily prepared in three steps, as previously published, starting from 4- piperidone. The synthesis presented in this paper could be of interest for heterocyclic and general organic chemistry. The newly designed compounds possessing two pharmacophores, opioid and D2/D3, are potentially useful pharmacological probes. Of particular interest would be the simultaneous binding to both opioid and D2/D3 receptors, and the resulting pharmacological responses may be useful for the further understanding of tolerance and dependence phenomena in opioid clinical use and/or abuse.

Design and Evaluation of Heterobivalent PAR1-PAR2 Ligands as Antagonists of Calcium Mobilization.

A novel class of bivalent ligands targeting putative protease-activated receptor (PAR) heteromers has been prepared based upon reported antagonists for the subtypes PAR1 and PAR2. Modified versions of the PAR1 antagonist RWJ-58259 containing alkyne adapters were connected via cycloaddition reactions to azide-capped polyethylene glycol (PEG) spacers attached to imidazopyridazine-based PAR2 antagonists. Initial studies of the PAR1-PAR2 antagonists indicated that they inhibited G alpha q-mediated calcium mobilization in endothelial and cancer cells driven by both PAR1 and PAR2 agonists. Compounds of this novel class hold promise for the prevention of restenosis, cancer cell metastasis, and other proliferative disorders.

Advances in Achieving Opioid Analgesia Without Side Effects.

Opioids are the most effective drugs for the treatment of severe pain, but they also cause addiction and overdose deaths, which have led to a worldwide opioid crisis. Therefore, the development of safer opioids is urgently needed. In this article, we provide a critical overview of emerging opioid-based strategies aimed at effective pain relief and improved side effect profiles. These approaches comprise biased agonism, the targeting of (i) opioid receptors in peripheral inflamed tissue (by reducing agonist access to the brain, the use of nanocarriers, or low pH-sensitive agonists); (ii) heteromers or multiple receptors (by monovalent, bivalent, and multifunctional ligands); (iii) receptor splice variants; and (iv) endogenous opioid peptides (by preventing their degradation or enhancing their production by gene transfer). Substantial advancements are underscored by pharmaceutical development of new opioids such as peripheral κ-receptor agonists, and by treatments augmenting the action of endogenous opioids, which have entered clinical trials. Additionally, there are several promising novel opioids comprehensively examined in preclinical studies, but also strategies such as biased agonism, which might require careful rethinking.