Bivalent Ligands Research Articles

Multivalent activation of GLP-1 and sulfonylurea receptors modulates β-cell second-messenger signaling and insulin secretion.

Linking two pharmacophores that bind different cell surface receptors into a single molecule can enhance cell-targeting specificity to cells that express the complementary receptor pair. In this report, we developed and tested a synthetic multivalent ligand consisting of glucagon-like peptide-1 (GLP-1) linked to glibenclamide (Glb) (GLP-1/Glb) for signaling efficacy in β-cells. Expression of receptors for these ligands, as a combination, is relatively specific to the β-cell in the pancreas. The multivalent GLP-1/Glb increased both intracellular cAMP and Ca2+, although Ca2+ responses were significantly depressed compared with the monomeric Glb. Moreover, GLP-1/Glb increased glucose-stimulated insulin secretion in a dose-dependent manner. However, unlike the combined monomers, GLP-1/Glb did not augment insulin secretion at nonstimulatory glucose concentrations in INS 832/13 β-cells or human islets of Langerhans. These data suggest that linking two binding elements, such as GLP-1 and Glb, into a single bivalent ligand can provide a unique functional agent targeted to β-cells.

Inhibition of EGFR Activation by Bivalent Ligands Based on a Cyclic Peptide Mimicking the Dimerization Arm Structure of EGFR.

The epidermal growth factor receptor (EGFR) is a receptor in the ErbB family, and is overexpressed in some cancer cells. Recent research has shown that, since clustering of the EGFR increases the possibility of its dimerization and activation, the dimerization state of the EGFR on the cell surface is important for the recognition of the EGFR. In case a bivalent inhibitor has an optimized linker length, the clusters of the EGFR could be recognized with high affinity and kinase activation, which depends on EGF, could be suppressed. Peptide 1, which is derived from the dimerization arm of the EGFR, has been found previously to inhibit autophosphorylation of the EGFR. In this study, bivalent ligands based on peptide 1 with linkers of poly(L-proline) or poly-[(glycine)4(L-serine)] have been designed and synthesized. Bivalent ligands with polyproline linkers could maintain the distance between the ligand moieties. The inhibitory activity of these bivalent ligands against EGFR autophosphorylation was measured and was found to increase as the linker enlarges up to a 15-mer proline linker. The inhibitory activity of a bivalent ligand 7b is significantly higher compared to the corresponding monomeric peptide 2a. This suggests that bivalent EGFR ligands with optimal and rigid linkers could recognize the clusters of the EGFR with higher affinity and suppress kinase activation involving EGF.

Bivalent Ligand UDCA-LPE Inhibits Pro-Fibrogenic Integrin Signalling by Inducing Lipid Raft-Mediated Internalization.

Ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE) is a synthetic bile acid-phospholipid conjugate with profound hepatoprotective and anti-fibrogenic functions in vitro and in vivo. Herein, we aimed to demonstrate the inhibitory effects of UDCA-LPE on pro-fibrogenic integrin signalling. UDCA-LPE treatment of human embryonic liver cell line CL48 and primary human hepatic stellate cells induced a non-classical internalization of integrin β1 resulting in dephosphorylation and inhibition of SRC and focal adhesion kinase (FAK). Signalling analyses suggested that UDCA-LPE may act as a heterobivalent ligand for integrins and lysophospholipid receptor1 (LPAR1) and co-immunoprecipitation demonstrated the bridging effect of UDCA-LPE on integrin β1 and LPAR1. The disruption of either the UDCA-moiety binding to integrins by RGD-containing peptide GRGDSP or the LPE-moiety binding to LPAR1 by LPAR1 antagonist Ki16425 reversed inhibitory functions of UDCA-LPE. The lack of inhibitory functions of UDCA-PE and UDCA-LPE derivatives (14:0 and 12:0, LPE-moiety containing shorter fatty acid chain) as well as the consistency of the translocation of UDCA-LPE and integrins, which co-fractionated with LPE but not UDCA, suggested that the observed UDCA-LPE-induced translocation of integrins was mediated by LPE endocytic transport pathway.

Design of a True Bivalent Ligand with Picomolar Binding Affinity for a G Protein-Coupled Receptor Homodimer.

Bivalent ligands have emerged as chemical tools to study G protein-coupled receptor dimers. Using a combination of computational, chemical, and biochemical tools, here we describe the design of bivalent ligand 13 with high affinity ( KDB1 = 21 pM) for the dopamine D2 receptor (D2R) homodimer. Bivalent ligand 13 enhances the binding affinity relative to monovalent compound 15 by 37-fold, indicating simultaneous binding at both protomers. Using synthetic peptides with amino acid sequences of transmembrane (TM) domains of D2R, we provide evidence that TM6 forms the interface of the homodimer. Notably, the disturber peptide TAT-TM6 decreased the binding of bivalent ligand 13 by 52-fold and had no effect on monovalent compound 15, confirming the D2R homodimer through TM6 ex vivo. In conclusion, by using a versatile multivalent chemical platform, we have developed a precise strategy to generate a true bivalent ligand that simultaneously targets both orthosteric sites of the D2R homodimer.

Synthesis toward Bivalent Ligands for the Dopamine D2 and Metabotropic Glutamate 5 Receptors.

In this study, we designed and synthesized heterobivalent ligands targeting heteromers consisting of the metabotropic glutamate 5 receptor (mGluR5) and the dopamine D2 receptor (D2R). Bivalent ligand 22a with a linker consisting of 20 atoms showed 4-fold increase in affinity for cells coexpressing D2R and mGluR5 compared to cells solely expressing D2R. Likewise, the affinity of 22a for mGluR5 increased 2-fold in the coexpressing cells. Additionally, 22a exhibited a 5-fold higher mGluR5 affinity than its monovalent precursor 21a in cells coexpressing D2R and mGluR5. These results indicate that 22a is able to bridge binding sites on both receptors constituting the heterodimer. Likewise, cAMP assays revealed that 22a had a 4-fold higher potency in stable D2R and mGluR5 coexpressing cell lines than 1. Furthermore, molecular modeling reveals that 22a is able to simultaneously bind both receptors by passing between the TM5-TM6 interface and establishing six protein-ligand H-bonds.

Identification of Bivalent Ligands with Melatonin Receptor Agonist and Fatty Acid Amide Hydrolase (FAAH) Inhibitory Activity That Exhibit Ocular Hypotensive Effect in the Rabbit.

Activation of melatonin receptors and inhibition of fatty acid amide hydrolase (FAAH) have both shown potential benefits for the treatment of glaucoma. To exploit the combination of these biological activities in single therapeutic agents, we designed dual-acting compounds sharing the pharmacophore elements required for the two targets, in search for balanced potencies as MT1/MT2 agonists and FAAH inhibitors. In particular, the N-anilinoethylamide scaffold, previously developed for melatonergic ligands, was decorated at meta position with a polymethylene linker bound to an O-arylcarbamate group, substituted according to known structure-activity relationships for FAAH inhibition. For the most active series, the N-anilinoethylamide portion was also replaced with the indole scaffold of melatonin. O-Biphenyl-3-ylcarbamate derivatives were characterized by remarkable and balanced activity at both targets, in the nanomolar range for compound 29. Topical administration reduced elevated intraocular pressure in rabbits, with a longer action and improved efficacy compared to the reference compounds melatonin and URB597.

Combined Glia Inhibition and Opioid Receptor Agonism Afford Highly Potent Analgesics without Tolerance.

Commonly prescribed opioid analgesics produce tolerance upon chronic use due in part to induction of hyperalgesia. Given that two reported bivalent ligands (MMG22 and MCC22) produce potent antinociception without tolerance only in inflamed mice, we have investigated the possible cellular and receptor targets of these ligands. The selective microglia inhibitors, minocycline and SB290157, antagonized intrathecal (i.t.) MCC22 antinociception orders of magnitude more potently than MMG22, suggesting that MCC22 selectively targets activated microglia. The astrocyte toxin, l-α-aminoadipic acid antagonized MMG22 antinociception 126-fold without reducing the potency of MCC22, indicating that activated astrocytes are targets of MMG22. MK-801 and Ro25-6981 antagonism of MMG22 antinociception, but not MCC22, is consistent with selective inhibition of activated NMDAR in astrocytes. The antinociception produced by i.t. MMG22 or MCC22 were both antagonized by the selective mu opioid receptor antagonist, β-FNA, implicating interaction of these ligands with MOR in spinal afferent neurons. MCC22 antinociception was potently blocked by kainate or AMPA ion channel antagonists (LY382884; NBQX), in contrast to MMG22. It is concluded that i.t. MMG22 and MCC22 produce exceptional antinociception via potent inhibition of activated spinal glia, thereby leading to desensitization of spinal neurons and enhanced activation of neuronal MOR. Thus, the present study suggests a new approach to treatment of chronic inflammatory pain without tolerance through a single molecular entity that simultaneously inhibits activated glia and stimulates MOR in spinal neurons.

A bivalent compound targeting CCR5 and the mu opioid receptor treats inflammatory arthritis pain in mice without inducing pharmacologic tolerance

BackgroundPain accompanies rheumatoid arthritis and other chronic inflammatory conditions and is difficult to manage. Although opioids provide potent analgesia, chronic opioid use can cause tolerance and addiction. Recent studies have demonstrated functional interactions between chemokine and opioid receptor signaling pathways. Reported heterodimerization of chemokine and opioid receptors led our group to develop bivalent compounds that bind both types of receptors, with the goal of targeting opioids to sites of inflammation. MCC22 is a novel bivalent compound containing a CCR5 antagonist and mu opioid receptor (MOR) agonist pharmacophores linked through a 22-atom spacer. We evaluated the efficacy of MCC22 in the K/B.g7 T-cell receptor transgenic mouse model of spontaneous inflammatory arthritis.MethodsMCC22 or morphine was administered intraperitoneally at varying doses to arthritic K/B.g7 mice or nonarthritic control mice. Mechanical pain hypersensitivity was measured each day before and after drug administration, using the electronic von Frey test. The potency of MCC22 relative to that of morphine was calculated. Functional readouts of pain included grip strength and nesting behavior. A separate dosing regimen was used to determine whether the drugs induced pharmacologic tolerance.ResultsMCC22 provided ~ 3000-fold more potent analgesia than morphine in this model. Daily treatment with MCC22 also led to a cumulative analgesic effect, reducing the daily baseline pain level. MCC22 produced no observable analgesic effect in nonarthritic control mice. Importantly, repeated administration of MCC22 did not induce pharmacologic tolerance, whereas a similar regimen of morphine did. Both grip strength and nesting behaviors improved among arthritic mice treated with MCC22. Ankle thickness and arthritis scores were not affected by MCC22. The analgesic effect of MCC22 was abolished in K/B.g7 mice genetically lacking CCR5, demonstrating the receptor specificity of the antagonist pharmacophore.ConclusionsMCC22 is a novel bivalent ligand that targets CCR5 and MOR. Our findings demonstrate that MCC22 provides highly potent analgesia and improved functional outcomes in a model of inflammatory arthritis, without inducing typical opioid tolerance. These findings suggest that MCC22 or similar compounds could be used to treat the pain associated with inflammatory arthritis and related conditions, while minimizing the risks typically associated with chronic opioid use.

Novel Bivalent and D-Peptide Ligands of CXCR4 Mobilize Hematopoietic Progenitor Cells to the Blood in C3H/HeJ Mice

The interaction of SDF-1α (also known as CXCL12) with the CXCR4 receptor plays a critical role in the retention of hematopoietic stem cells (HSCs) in bone marrow. The viral macrophage inflammatory protein-II (vMIP-II), a human herpesvirus-8 (HHV-8)-encoded viral chemokine, can bind the CXCR4 receptor and inhibit endogenous ligand-induced calcium responses and cell migration. Previously, we used the bivalent ligand approach to link synthetically two unnatural D-amino acid peptides derived from the N-terminus of vMIP-II (DV1 and DV3, respectively) to generate a dimeric peptide, DV1-K-(DV3) (also named HC4319), which shows very high affinity for CXCR4. Here, we studied the biological effects of this dimeric peptide, HC4319, and its monomeric counterpart, DV1, on SDF-1α-induced signaling in CXCR4- or CXCR7-transfected Chinese hamster ovary cells and mobilization of hematopoietic progenitor cells (HPCs) in C3H/HeJ mice using an HPC assay. HC4319 and DV1 inhibited significantly the phosphorylation of Akt and Erk, known to be downstream signaling events of CXCR4. This in vivo study in C3H/HeJ mice showed that HC4319 and DV-1 strongly induced rapid mobilization of granulocyte–macrophage colony-forming units (CFUs), erythrocyte burst-forming units, and granulocyte–erythrocyte–monocyte–megakaryocyte CFUs from the bone marrow to the blood. These results provide the first reported experimental evidence that bivalent and D-amino acid peptides derived from the N-terminus of vMIP-II are potent mobilizers of HPCs in C3H/HeJ mice and support the further development of such agents for clinical application.

Target-Directed Self-Assembly of Homodimeric Drugs Against β-Tryptase.

Tryptase, a serine protease released from mast cells, is implicated in many allergic and inflammatory disorders. Human tryptase is a donut-shaped tetramer with the active sites facing inward forming a central pore. Bivalent ligands spanning two active sites potently inhibit this configuration, but these large compounds have poor drug-like properties. To overcome some of these challenges, we developed self-assembling molecules, called coferons, which deliver a larger compound in two parts. Using a pharmacophoric core and reversibly binding linkers to span two active sites, we have successfully produced three novel homodimeric tryptase inhibitors. Upon binding to tryptase, compounds reassembled into flexible homodimers, with significant improvements in IC50 (0.19 ± 0.08 μM) over controls (5.50 ± 0.09 μM), and demonstrate good activity in mast cell lines. These studies provide validation for this innovative technology that is especially well-suited for the delivery of dimeric drugs to modulate intracellular macromolecular targets.

Abstract 1671: Identification and characterization of MAM03055A, a novel bivalent sigma-2 receptor agonist

Abstract Sigma-2 receptors are highly expressed in various types of cancer and mediate dual effects on cancer cell viability. Agonists induce programmed cell death by a number of pathways including apoptosis and autophagy. Other classes of sigma-2 receptor ligands activate pathways related to glycolytic metabolism and hypoxia. CM572 is a 6-isothiocyanato benzoxazolone derivative that binds irreversibly and selectively to sigma-2 receptors, presumably through attack of the isothiocyanate moiety by a receptor nucleophile. It is a partial agonist, inducing dose-dependent cell death. We observed by LC/MS analysis, that upon storage in DMSO solution at -20oC over several months, CM572 degrades into a mixture of compounds that appear to retain both receptor binding activity and ability to induce cell death. The degradation was eventually convergent to one main compound (CM572-HN). CM572-HN was identified as a dimeric molecule based on the isotopic fingerprint of the high-resolution mass spectrum. Similarly, a degradation study by 1H NMR of a solution of CM572 in deuterated DMSO showed convergence to one major compound. The degradation site was narrowed down to the isothiocyanate group, as only the signals for its neighboring protons were shifted in the degradation product. We suspected hydrolysis, the final degradation step thus being the corresponding amine. It was then hypothesized that the kinetics of degradation of CM572 towards the corresponding 6-substituted amine (CM571) was slower than that of the nucleophilic attack of CM571 on the isothiocyanate group of CM572. Hence, CM571 and CM572 were synthesized and then reacted together to produce, in fact, a single compound (MAM03055A), with mass identical to one originally identified in the stock solution and 1H NMR spectra fitting those of the compound originally obtained during the NMR degradation study. Thus the novel compound is essentially a thiourea-linked dimer of the amine, CM571. Receptor binding assays revealed that MAM03055A (MAM) exhibited selectivity for sigma-2 receptors over sigma-1, with sigma-1 Ki = 2,829 nM and sigma-2 Ki = 56.7 nM, resulting in 50-fold selectivity for sigma-2 receptors. MAM induced dose-dependent cell death in human SK-N-SH neuroblastoma cells, with an EC50 = 9.24 ± 1.15 µM for a 24h incubation. Interestingly, unlike other sigma-2 agonists that give 100% cell kill, MAM reached a maximum of only 80%, even at a concentration of 100 µM. The true EC50 in this case was therefore around 2 µM. Thus, MAM behaved like a partial agonist. The binding affinities and cytotoxic activity of the dimer resembles that of CM572 (isothiocyanate; EC50 = 7.6 µM), but is very different from CM571 (amine), which has high affinity at both subtypes and stimulates metabolism. Bivalent sigma-2 ligands may thus have interesting properties that deserve further exploration. (*C. L. and M. M. contributed equally as first authors) Citation Format: Cheri Z. Liu, Marco Mottinelli, Hilary E. Nicholson, Eric Zhong, Christopher R. McCurdy, Wayne D. Bowen. Identification and characterization of MAM03055A, a novel bivalent sigma-2 receptor agonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1671.

The size matters? A computational tool to design bivalent ligands.

MotivationBivalent ligands are increasingly important such as for targeting G protein-coupled receptor (GPCR) dimers or proteolysis targeting chimeras (PROTACs). They contain two pharmacophoric units that simultaneously bind in their corresponding binding sites, connected with a spacer chain. Here, we report a molecular modelling tool that links the pharmacophore units via the shortest pathway along the receptors van der Waals surface and then scores the solutions providing prioritization for the design of new bivalent ligands.ResultsBivalent ligands of known dimers of GPCRs, PROTACs and a model bivalent antibody/antigen system were analysed. The tool could rapidly assess the preferred linker length for the different systems and recapitulated the best reported results. In the case of GPCR dimers the results suggest that in some cases these ligands might bind to a secondary binding site at the extracellular entrance (vestibule or allosteric site) instead of the orthosteric binding site.Availability and implementationFreely accessible from the Molecular Operating Environment svl exchange server (https://svl.chemcomp.com/).Supplementary information Supplementary data are available at Bioinformatics online.

A Y-Shaped Three-Arm Structure for Probing Bivalent Interactions between Protein Receptor–Ligand Using AFM and SPR

The goal of this research was to develop linkage chemistry for the study of bivalent interactions between a receptor and its ligand using atomic force microscopy (AFM) and surface plasmon resonance (SPR). We conceived a three-arm structure composed of flexible chains connected to a large rigid core with orthogonal functional groups at their ends for formation and attachment (or immobilization) of bivalent ligands. To demonstrate the principle, we chose the well-known biotin-streptavidin interaction as a model system. On the basis of a crystal structure of the biotin-streptavidin complex, we designed and synthesized a bisbiotin ligand to have a Y shape with two biotin motifs on its arms for binding and a functional group on its stem for immobilization or attachment, referred to as y-bisbiotin. First, we found that the y-bisbiotin ligand stabilized the streptavidin more than its monobiotin counterpart did in solution, which indicates that the bivalent interaction was synergistic. The y-bisbiotin was attached to AFM tips through a click reaction for the force measurement experiments, which showed that unbinding the bisbiotin from streptavidin needed twice the force of unbinding a monobiotin. For the SPR study, we added a ω-thiolated alkyl chain to y-bisbiotin for its incorporation into a monolayer. The SPR data indicated that the streptavidin dissociated from a mixed monolayer bearing y-bisbiotin much slower than from the one bearing monobiotin. This work demonstrates unique chemistry for the study of bivalent interactions using AFM and SPR.

CLEC10A Is a Specific Marker for Human CD1c+ Dendritic Cells and Enhances Their Toll-Like Receptor 7/8-Induced Cytokine Secretion.

Dendritic cells (DCs) are major players for the induction of immune responses. Apart from plasmacytoid DCs (pDCs), human DCs can be categorized into two types of conventional DCs: CD141+ DCs (cDC1) and CD1c+ DCs (cDC2). Defining uniquely expressed surface markers on human immune cells is not only important for the identification of DC subpopulations but also a prerequisite for harnessing the DC subset-specific potential in immunomodulatory approaches, such as antibody-mediated antigen targeting. Although others identified CLEC9A as a specific endocytic receptor for CD141+ DCs, such a receptor for CD1c+ DCs has not been discovered, yet. By performing transcriptomic and flow cytometric analyses on human DC subpopulations from different lymphohematopoietic tissues, we identified CLEC10A (CD301, macrophage galactose-type C-type lectin) as a specific marker for human CD1c+ DCs. We further demonstrate that CLEC10A rapidly internalizes into human CD1c+ DCs upon binding of a monoclonal antibody directed against CLEC10A. The binding of a CLEC10A-specific bivalent ligand (the MUC-1 peptide glycosylated with N-acetylgalactosamine) is limited to CD1c+ DCs and enhances the cytokine secretion (namely TNFα, IL-8, and IL-10) induced by TLR 7/8 stimulation. Thus, CLEC10A represents not only a candidate to better define CD1c+ DCs—due to its high endocytic potential—CLEC10A also exhibits an interesting candidate receptor for future antigen-targeting approaches.

A constraint scaffold enhances affinity of a bivalent N-acetylglucosamine ligand against wheat germ agglutinin

Bivalent glycoconjugates have a minimal valence with avidity potential on protein-carbohydrate interactions as well as simplicity of chemical structures enabling simple synthesis with low cost. Understanding the way to maximize the affinities of bivalent glycoconjugates is important for the development of cost-effective tools for therapeutic and diagnostic research. However, there has been little discussion about the effects of constraints imposed from ligand scaffolds on the binding abilities. We synthesized three kinds of biantennary N-acetylglucosamine glycosides with different scaffolds using isobutenyl bis(propargyl)ether as a common scaffold precursor. Decoration of the scaffold branches with GlcNAc moieties through copper-catalyzed azide-alkyne cycloaddition and grafting of the alkenyl focal point to another bivalent biotin dendron through thiol-ene and nucleophilic substitution reactions were successfully carried out in an orthogonal manner. The association constants of the ligands against wheat germ agglutinin were determined by a fluorometric titration assay. A bivalent biotin counterpart provided higher affinity than an isobutyl scaffold, whereas an isobutenyl scaffold yielded more enhancement than a bivalent biotin counterpart. The present work suggested that the constraint and steric bulk of ligand scaffolds are possible factors for improving binding properties of glycoconjugates against lectins or proteins.

Developing a Biased Unmatched Bivalent Ligand (BUmBL) Design Strategy to Target the GPCR Homodimer Allosteric Signaling (cAMP over β-Arrestin 2 Recruitment) Within the Melanocortin Receptors.

Understanding the functional relevance of G protein-coupled receptor (GPCR) homodimerization has been limited by the insufficient tools to assess asymmetric signaling occurring within dimers comprised of the same receptor type. We present unmatched bivalent ligands (UmBLs) to study the asymmetric function of melanocortin homodimers. UmBLs contain one agonist and one antagonist pharmacophore designed to target a melanocortin homodimer such that one receptor is occupied by an agonist and the other receptor by an antagonist pharmacophore. First-in-class biased UmBLs (BUmBLs) targeting the human melanocortin-4 receptor (hMC4R) were discovered. The BUmBLs displayed biased agonism by potently stimulating cAMP signaling (EC50 ∼ 2-6 nM) but minimally activating the β-arrestin recruitment pathway (≤55% maximum signal at 10 μM). To our knowledge, we report the first single-compound strategy to pharmacologically target melanocortin receptor allosteric signaling that occurs between homodimers that can be applied straightforwardly in vitro and in vivo to other GPCR systems.

Design, Synthesis, and Biological Evaluation of Bivalent Ligands Targeting Dopamine D2 -Like Receptors and the μ-Opioid Receptor.

Currently, there is mounting evidence that intermolecular receptor-receptor interactions may result in altered receptor recognition, pharmacology and signaling. Heterobivalent ligands have been proven useful as molecular probes for confirming and targeting heteromeric receptors. This report describes the design and synthesis of novel heterobivalent ligands for dopamine D2 -like receptors (D2 -likeR) and the μ-opioid receptor (μOR) and their evaluation using ligand binding and functional assays. Interestingly, we identified a potent bivalent ligand that contains a short 18-atom linker and combines good potency with high efficacy both in β-arrestin 2 recruitment for μOR and MAPK-P for D4 R. Furthermore, this compound was characterized by a biphasic competition binding curve for the D4 R-μOR heterodimer, indicative of a bivalent binding mode. As this compound possibly bridges the D4 R-μOR heterodimer, it could be used as a pharmacological tool to further investigate the interactions of D4 R and μOR.

Enhancing β‐cell Sensitivity to Kisspeptin via Linkage to GLP‐1

Glucagon Like Peptide‐1 (GLP‐1) and Kisspeptin (KP) receptors are co‐expressed on pancreatic β‐cells, and activation of both receptors potentiate glucose stimulated insulin secretion (GSIS), although they appear to work via different downstream signaling pathways. Unlike GLP‐1, KP has a low affinity for receptor activation (EC50 20 nM vs >1 uM) making it difficult to use as a drug without significant side effects. We developed a bivalent ligand composed of GLP‐17–36 and KP‐10 binding domains linked together, to evaluate if linking KP to a high affinity ligand would promote its activity at low concentrations by presumably increasing its local membrane concentration after GLP‐1 binding. This feature may also provide some specificity for targeting to β‐cells which express both receptors. The effects of KP‐10, GLP‐1 and the KP‐10/GLP‐1 bivalent analog on insulin secretion from the INS 832/3 cell line were investigated. KP‐10 by itself had no effect on insulin secretion in the absence of glucose, but more than doubled GSIS (basal, 0.23 +/− 0.07; GSIS 0.60 +/− 0.02 ng/min) with an EC50 of ~25 μM. At saturating doses, KP‐10 (100 μm) and GLP‐1 (100 nM) had similar effects on GSIS, and together as monomers had an additive effect (0.79 +/− 0.07 (ng/min) consistent with activation of independent downstream signaling pathways. The dimer also did not activate secretion in the absence of glucose, but doubled GSIS much like the individual monomers (0.64 +/− 0.01 ng/min); that is, an additive response was not obvious. This finding suggests that either the KP‐10 moiety is not binding or is not active in the bivalent ligand, or that GLP‐1 activity is diminished in the bivalent construct such that the combined effect of GLP‐1/KP‐10 is diminished when compared to the additive effect or the monomers. We have evidence from previous studies that binding of the GLP‐1 moiety within a bivalent construct is decreased relative to monomeric GLP‐11, suggesting an additive response may be present. Currently, we are determining the ability of GLP‐1/KP‐10 to initiate downstream signaling from both ligands to resolve the question: is KP active in the GLP‐1/KP at concentrations where monomeric KP‐10 is not.Support or Funding InformationFunded by the American Diabetes Association and Juvenile Diabetes Research FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bivalent ligand MCC22 potently attenuates nociception in a murine model of sickle cell disease.

Sickle cell disease (SCD) is a chronic inflammatory disorder accompanied by chronic pain. In addition to ongoing pain and hyperalgesia, vaso-occlusive crises-induced pain can be chronic or episodic. Because analgesics typically used to treat pain are not very effective in SCD, opioids, including morphine, are a primary treatment for managing pain in SCD but are associated with many serious side effects, including constipation, tolerance, addiction, and respiratory depression. Thus, there is a need for the development of novel treatments for pain in SCD. In this study, we used the Townes transgenic mouse model of SCD to investigate the antinociceptive efficacy of the bivalent ligand, MCC22, and compared its effectiveness with morphine. MCC22 consists of a mu-opioid receptor agonist and a chemokine receptor-5 (CCR5) antagonist that are linked through a 22-atom spacer. Our results show that intraperitoneal administration of MCC22 produced exceptionally potent dose-dependent antihyperalgesia as compared to morphine, dramatically decreased evoked responses of nociceptive dorsal horn neurons, and decreased expression of proinflammatory cytokines in the spinal cord. Moreover, tolerance did not develop to its analgesic effects after repeated administration. In view of the extraordinary potency of MCC22 without tolerance, MCC22 and similar compounds may vastly improve the management of pain associated with SCD.

Design of Bivalent Nucleic Acid Ligands for Recognition of RNA-Repeated Expansion Associated with Huntington's Disease.

We report the development of a new class of nucleic acid ligands that is comprised of Janus bases and the MPγPNA backbone and is capable of binding rCAG repeats in a sequence-specific and selective manner via, inference, bivalent H-bonding interactions. Individually, the interactions between ligands and RNA are weak and transient. However, upon the installation of a C-terminal thioester and an N-terminal cystine and the reduction of disulfide bond, they undergo template-directed native chemical ligation to form concatenated oligomeric products that bind tightly to the RNA template. In the absence of an RNA target, they self-deactivate by undergoing an intramolecular reaction to form cyclic products, rendering them inactive for further binding. The work has implications for the design of ultrashort nucleic acid ligands for targeting rCAG-repeat expansion associated with Huntington's disease and a number of other related neuromuscular and neurodegenerative disorders.