cAMP Accumulation Research Articles

Immunomodulatory Effects of Fluoroquinolones in Community-Acquired Pneumonia-Associated Acute Respiratory Distress Syndrome.

Community-acquired pneumonia is reported as one of the infectious diseases that leads to the development of acute respiratory distress syndrome. The innate immune system is the first line of defence against microbial invasion; however, its dysregulation during infection, resulting in an increased pathogen load, stimulates the over-secretion of chemokines and pro-inflammatory cytokines. This phenomenon causes damage to the epithelial-endothelial barrier of the pulmonary alveoli and the leakage of the intravascular protein into the alveolar lumen. Fluoroquinolones are synthetic antimicrobial agents with immunomodulatory properties that can inhibit bacterial proliferation as well as exhibit anti-inflammatory activities. It has been demonstrated that the structure of fluoroquinolones, particularly those with a cyclopropyl group, exerts immunomodulatory effects. Its capability to inhibit phosphodiesterase activity leads to the accumulation of intracellular cAMP, which subsequently enhances PKA activity, resulting in the inhibition of transcriptional factor NF-κB and the activation of CREB. Another mechanism reported is the inhibition of TLR and ERK signalling pathways. Although the sequence of events has not been completely understood, significant progress has been made in comprehending the specific mechanisms underlying the immunomodulatory effects of fluoroquinolones. Here, we review the indirect immunomodulatory effects of FQs as an alternative to empirical therapy in patients diagnosed with community-acquired pneumonia.

Abstract 5933: Identifying MC4R-targeted small molecule to investigate MC4R impact on cachexia progression

Abstract Background: The melanocortin system participates in energy homeostasis and appetiteregulation. This metabolic system is primarily located in the central nervous system,specifically in the hypothalamus and brainstem region. Within the melanocortin system,there are five established melanocortin receptors (MCR). MCRs are g-protein-coupledreceptors (GPCRs) and when these receptors are activated, they can increase neuronexcitation, regulate neurotransmitter release, regulate synaptic input, and alterconnection and strength. When Melanocortin-4 Receptor (MC4R) plays a critical role inthe progression of multiple metabolic diseases including cachexia. Cachexia is definedas the reduction of fat and lean mass, which is caused not just by lack of appetite, but byinflammatory impact. Cachexia is a symptom seen in numerous chronic diseases fromcancer to mitochondrial dysfunction to autoimmune inflictions. This makes MC4R apotentially strong therapeutic target. Method: In-silico and high-throughput methods were used to identify small moleculesthat target and inhibit MC4R. For high-throughput screen, Hek293 cells withconstitutively active MC4R tagged with a nanoluciferase were used to identify molecules,specifically with inhibitory qualities. cAMP accumulation and β-arrestin recruitmentassay were used to determine the impact on MC4R functionality. Results: 4738 small molecules were screened using high-throughput methods. Using a60% cutoff, 21 small molecules were identified as potential MC4R inhibitors. 17 arecurrently under investigation to confirm high-throughput results and identify functionalimpact. Several of these molecules have shown promising potential in vitro and will likelymove forward into in vivo studies. Conclusion: This study identifies prospective small molecule inhibitors that targetMC4R and may be used as a probe to explore MC4R biology. Citation Format: Julia Stokes, Dhanir Taylor, Marie Foss, Benedikt Grau, Sanjay Malhotra. Identifying MC4R-targeted small molecule to investigate MC4R impact on cachexia progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5933.

AKAP12 Upregulation Associates With PDE8A to Accelerate Cardiac Dysfunction.

In heart failure, signaling downstream the β2-adrenergic receptor is critical. Sympathetic stimulation of β2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind β2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. cAMP accumulation in real time downstream of the β2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.

Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of Gs Protein to the Melatonin MT1 Receptor.

Melatonin is a neuroendocrine hormone that regulates the circadian rhythm and many other physiological processes. Its functions are primarily exerted through two subtypes of human melatonin receptors, termed melatonin type-1 (MT1) and type-2 (MT2) receptors. Both MT1 and MT2 receptors are generally classified as Gi-coupled receptors owing to their well-recognized ability to inhibit cAMP accumulation in cells. However, it remains an enigma as to why melatonin stimulates cAMP production in a number of cell types that express the MT1 receptor. To address if MT1 can dually couple to Gs and Gi proteins, we employed a highly sensitive luminescent biosensor (GloSensorTM) to monitor the real-time changes in the intracellular cAMP level in intact live HEK293 cells that express MT1 and/or MT2. Our results demonstrate that the activation of MT1, but not MT2, leads to a robust enhancement on the forskolin-stimulated cAMP formation. In contrast, the activation of either MT1 or MT2 inhibited cAMP synthesis driven by the activation of the Gs-coupled β2-adrenergic receptor, which is consistent with a typical Gi-mediated response. The co-expression of MT1 with Gs enabled melatonin itself to stimulate cAMP production, indicating a productive coupling between MT1 and Gs. The possible existence of a MT1-Gs complex was supported through molecular modeling as the predicted complex exhibited structural and thermodynamic characteristics that are comparable to that of MT1-Gi. Taken together, our data reveal that MT1, but not MT2, can dually couple to Gs and Gi proteins, thereby enabling the bi-directional regulation of adenylyl cyclase to differentially modulate cAMP levels in cells that express different complements of MT1, MT2, and G proteins.

The regulatory effects of (p)ppGpp and indole on cAMP synthesis in Escherichia coli cells.

Bacterial stress adaptive response is formed due to changes in the cell gene expression profile in response to alterations in environmental conditions through the functioning of regulatory networks. The mutual influence of network signaling molecules represented by cells' natural metabolites, including indole and second messengers (p) ppGpp and cAMP, is hitherto not well understood, being the aim of this study. E. coli parent strain BW25141 ((p) ppGpp+) and deletion knockout BW25141ΔrelAΔspoT which is unable to synthesize (p)ppGpp ((p)ppGpp0) were cultivated in M9 medium supplemented with different glucose concentrations (5.6 and 22.2 mM) in the presence of tryptophan as a substrate for indole synthesis and in its absence. The glucose content was determined with the glucose oxidase method; the indole content, by means of HPLC; and the cAMP concentration, by ELISA. The onset of an increase in initially low intracellular cAMP content coincided with the depletion of glucose in the medium. Maximum cAMP accumulation in the cells was proportional to the concentration of initially added glucose. At the same time, the (p) ppGpp0 mutant showed a decrease in maximum cAMP levels compared to the (p)ppGpp+ parent, which was the most pronounced in the medium with 22.2 mM glucose. So, (p)ppGpp was able to positively regulate cAMP formation. The promoter of the tryptophanase operon responsible for indole biosynthesis is known to be under the positive control of catabolic repression. Therefore, in the cells of the (p)ppGpp+ strain grown in the tryptophan-free medium that were characterized by a low rate of spontaneous indole formation, its synthesis significantly increased in response to the rising cAMP level just after glucose depletion. However, this was not observed in the (p)ppGpp0 mutant cells with reduced cAMP accumulation. When tryptophan was added to the medium, both of these strains demonstrated high indole production, which was accompanied by a decrease in cAMP accumulation compared to the tryptophan-free control. Thus, under glucose depletion, (p)ppGpp can positively regulate the accumulation of both cAMP and indole, while the latter, in its turn, has a negative effect on cAMP formation.

Identification and functional analysis of first heterozygous frameshift mutation in the GHRH gene in a Chinese boy with isolated growth hormone deficiency

BackgroundIsolated growth hormone deficiency (IGHD) is a rare genetically heterogeneous disorder caused primarily by mutations in GH1 and GH releasing hormone receptor (GHRHR). The aim of this study was to identify the molecular etiology of a Chinese boy with IGHD. MethodsWhole-exome sequencing, sanger sequencing and bioinformatic analysis were performed to screen for candidate mutations. The impacts of candidate mutation on gene expression, intracellular localization and protein function were further evaluated by in vitro assays. ResultsA novel heterozygous frameshift mutation in the GHRH gene (c.91dupC, p.R31Pfs*98) was identified in a Chinese boy clinically diagnosed as having IGHD. The mutation was absent in multiple public databases, and considered as deleterious using in silico prediction, conservative analysis and three-dimensional homology modeling. Furthermore, mRNA and protein expression levels of mutant GHRH were significantly increased than wild-type GHRH (p < 0.05). Moreover, mutant GHRH showed an aberrant accumulation within the cytoplasm, and obviously reduced ability to stimulate GH secretion and cAMP accumulation in human GHRHR-expressing pituitary GH3 cells compared to wild-type GHRH (p < 0.05). ConclusionOur study discovered the first loss-of function mutation of GHRH in a Chinese boy with IGHD and provided new insights on IGHD pathogenesis caused by GHRH haploinsufficiency.

ADAM19 cleaves the PTH receptor and associates with brachydactyly type E.

Brachydactyly type E (BDE), shortened metacarpals, metatarsals, cone-shaped epiphyses, and short stature commonly occurs as a sole phenotype. Parathyroid hormone-like protein (PTHrP) has been shown to be responsible in all forms to date, either directly or indirectly. We used linkage and then whole genome sequencing in a small pedigree, to elucidate BDE and identified a truncated disintegrin-and-metalloproteinase-19 (ADAM19) allele in all affected family members, but not in nonaffected persons. Since we had shown earlier that the extracellular domain of the parathyroid hormone receptor (PTHR1) is subject to an unidentified metalloproteinase cleavage, we tested the hypothesis that ADAM19 is a sheddase for PTHR1. WT ADAM19 cleaved PTHR1, while mutated ADAM-19 did not. We mapped the cleavage site that we verified with mass spectrometry between amino acids 64-65. ADAM-19 cleavage increased Gq and decreased Gs activation. Moreover, perturbed PTHR1 cleavage by ADAM19 increased ß-arrestin2 recruitment, while cAMP accumulation was not altered. We suggest that ADAM19 serves as a regulatory element for PTHR1 and could be responsible for BDE. This sheddase may affect other PTHrP or PTH-related functions.

Role of G protein-coupled receptor kinases (GRKs) in β2 -adrenoceptor-mediated glucose uptake.

Truncation of the C-terminal tail of the β2 -AR, transfection of βARKct or over-expression of a kinase-dead GRK mutant reduces isoprenaline-stimulated glucose uptake, indicating that GRK is important for this response. We explored whether phosphorylation of the β2 -AR by GRK2 has a role in glucose uptake or if this response is related to the role of GRK2 as a scaffolding protein. CHO-GLUT4myc cells expressing wild-type and mutant β2 -ARs were generated and receptor affinity for [3 H]-CGP12177A and density of binding sites determined together with the affinity of isoprenaline and BRL37344. Following receptor activation by β2 -AR agonists, cAMP accumulation, GLUT4 translocation, [3 H]-2-deoxyglucose uptake, and β2 -AR internalization were measured. Bioluminescence resonance energy transfer was used to investigate interactions between β2 -AR and β-arrestin2 or between β2 -AR and GRK2. Glucose uptake after siRNA knockdown or GRK inhibitors was measured in response to β2 -AR agonists. BRL37344 was a poor partial agonist for cAMP generation but displayed similar potency and efficacy to isoprenaline for glucose uptake and GLUT4 translocation. These responses to β2 -AR agonists occurred in CHO-GLUT4myc cells expressing β2 -ARs lacking GRK or GRK/PKA phosphorylation sites as well as in cells expressing the wild-type β2 -AR. However, β2 -ARs lacking phosphorylation sites failed to recruit β-arrestin2 and did not internalize. GRK2 knock-down or GRK2 inhibitors decreased isoprenaline-stimulated glucose uptake in rat L6 skeletal muscle cells. Thus, GRK phosphorylation of the β2 -AR is not associated with isoprenaline- or BRL37344-stimulated glucose uptake. However, GRKs acting as scaffold proteins are important for glucose uptake as GRK2 knock-down or GRK2 inhibition reduces isoprenaline-stimulated glucose uptake.

Novel 1-(1-Arylimiazolin-2-Yl)-3-Arylalkilurea Derivatives with Modulatory Activity on Opioid MOP Receptors.

μ-opioid receptor ligands such as morphine and fentanyl are the most known and potent painkillers. However, the severe side effects seen with their use significantly limit their widespread use. The continuous broadening of knowledge about the properties of the interactions of the MOP receptor (human mu opioid receptor, OP3) with ligands and specific intracellular signaling pathways allows for the designation of new directions of research with respect to compounds with analgesic effects in a mechanism different from classical ligands. Allosteric modulation is an extremely promising line of research. Compounds with modulator properties may provide a safer alternative to the currently used opioids. The aim of our research was to obtain a series of urea derivatives of 1-aryl-2-aminoimidazoline and to determine their activity, mechanism of biological action and selectivity toward the MOP receptor. The obtained compounds were subjected to functional tests (cAMP accumulation and β-arrestin recruitment) in vitro. One of the obtained compounds, when administered alone, did not show any biological activity, while when co-administered with DAMGO, it inhibited β-arrestin recruitment. These results indicate that this compound is a negative allosteric modulator (NAM) of the human MOP receptor.

Epidural administration of large dose of opioid μ receptor agonist may impair cardiac functions and myocardial viability via desensitizing transient receptor potential vanilloid 1

The incidence of postoperative myocardial injury remains high as the underlying pathogenesis is still unknown. The dorsal root ganglion (DRG) neurons express transient receptor potential vanilloid 1 (TRPV1) and its downstream effector, calcitonin gene-related peptide (CGRP) participating in transmitting pain signals and cardiac protection. Opioids remain a mainstay therapeutic option for moderate-to-severe pain relief clinically, as a critical component of multimodal postoperative analgesia via intravenous and epidural delivery. Evidence indicates the interaction of opioids and TRPV1 activities in DRG neurons. Here, we verify the potential impairment of myocardial viability by epidural usage of opioids in postoperative analgesia. We found that large dose of epidural morphine (50 μg) significantly worsened the cardiac performance (+dP/dtmax reduction by 11% and -dP/dtmax elevation by 24%, all P < 0.001), the myocardial infarct size (morphine vs Control, 0.54 ± 0.09 IS/AAR vs. 0.23 ± 0.06 IS/AAR, P < 0.001) and reduced CGRP in the myocardium (morphine vs. Control, 9.34 ± 2.24 pg/mg vs. 21.23 ± 4.32 pg/mg, P < 0.001), while induced definite suppression of nociception in the postoperative animals. It was demonstrated that activation of μ-opioid receptor (μ-OPR) induced desensitization of TRPV1 by attenuating phosphorylation of the channel in the dorsal root ganglion neurons, via inhibiting the accumulation of cAMP. CGRP may attenuated the buildup of ROS and the reduction of mitochondrial membrane potential in cardiomyocytes induced by hypoxia/reoxygenation. The findings of this study indicate that epidurally giving large dose of μ-OPR agonist may aggravate myocardial injury by inhibiting the activity of TRPV1/CGRP pathway.

Pharmacologic characterization of atogepant: A potent and selective calcitonin gene-related peptide receptor antagonist.

The trigeminal sensory neuropeptide calcitonin gene-related peptide (CGRP) is identified as an essential element in migraine pathogenesis. In vitro and in vivo studies evaluated pharmacologic properties of the CGRP receptor antagonist atogepant. Radioligand binding using 125I-CGRP and cyclic adenosine monophosphate (cAMP) accumulation assays were conducted in human embryonic kidney 293 cells to assess affinity, functional potency and selectivity. Atogepant in vivo potency was assessed in the rat nitroglycerine model of facial allodynia and primate capsaicin-induced dermal vasodilation (CIDV) pharmacodynamic model. Cerebrospinal fluid/brain penetration and behavioral effects of chronic dosing and upon withdrawal were evaluated in rats. Atogepant exhibited high human CGRP receptor-binding affinity and potently inhibited human α-CGRP-stimulated cAMP responses. Atogepant exhibited significant affinity for the amylin1 receptor but lacked appreciable affinities for adrenomedullin, calcitonin and other known neurotransmitter receptor targets. Atogepant dose-dependently inhibited facial allodynia in the rat nitroglycerine model and produced significant CIDV inhibition in primates. Brain penetration and behavioral/physical signs during chronic dosing and abrupt withdrawal were minimal in rats. Atogepant is a competitive antagonist with high affinity, potency and selectivity for the human CGRP receptor. Atogepant demonstrated a potent, concentration-dependent exposure/efficacy relationship between atogepant plasma concentrations and inhibition of CGRP-dependent effects.

Dopamine D3 receptor modulates D2 receptor effects on cAMP and GABA release at striatopallidal terminals-Modulation by the Ca2+-Calmodulin-CaMKII system.

Dopamine D2 receptor (D2R) is expressed in striatopallidal neurons and decreases forskolin-stimulated cyclic adenine monophosphate (cAMP) accumulation and gamma-aminobutyric acid (GABA) release. Dopamine D3 receptor (D3R) mRNA is expressed in a population of striatal D2R-expressing neurons. Also, D3R protein and binding have been reported in the neuropil of globus pallidus. We explore whether D2R and D3R colocalize in striatopallidal terminals and whether D3R modulates the D2R effect on forskolin-stimulated [3H]cAMP accumulation in pallidal synaptosomes and high K+ stimulated-[3H]GABA release in pallidal slices. Previous reports in heterologous systems indicate that calmodulin (CaM) and CaMKII modulate D2R and D3R functions; thus, we study whether this system regulates its functional interaction. D2R immunoprecipitates with CaM, and pretreatment with ophiobolin A or depolarization of synaptosomes with 15 mM of K+ decreases it. Both treatments increase the D2R inhibition of forskolin-stimulated [3H]cAMP accumulation when activated with quinpirole, indicating a negative modulation of CaM on D2R function. Quinpirole also activates D3R, potentiating D2R inhibition of cAMP accumulation in the ophiobolin A-treated synaptosomes. D2R and D3R immunoprecipitate in pallidal synaptosomes and decrease after the kainic acid striatal lesion, indicating the striatal origin of the presynaptic receptors. CaM-kinase II alfa (CaMKIIα) immunoprecipitates with D3R and increases after high K+ depolarization. In the presence of KN62, a CaMKIIα blocker, D3R potentiates D2R effects on cAMP accumulation in depolarized synaptosomes and GABA release in pallidal slices, indicating D3R function regulation by CaMKIIα. Our data indicate that D3R potentiates the D2R effect on cAMP accumulation and GABA release at pallidal terminals, an interaction regulated by the CaM-CaMKIIα system.

Low-dose imiquimod induces melanogenesis in melanoma cells through an ROS-mediated pathway

BackgroundMelanogenesis is the process of melanin maturation which not only protects skin from UV radiation but also plays an important role in antigenicity of melanomas. Imiquimod (IMQ) is a toll-like receptor 7 (TLR7) agonist that exhibits antiviral and anticancer activity. ObjectiveTo explore whether IMQ could induce melanogenesis in melanoma cells. MethodsThe mouse melanoma cell line B16F10, the mouse immortalized melanocyte Melan-A, and human melanoma cell lines MNT-1, C32 and A375 were utilized in this study. The pigmented level was observed by the centrifuged cell pellet. The intracellular and extracellular melanin levels were examined in the absorbance in NaOH-extracted cell lysate and cell-cultured medium, respectively. The expression of melanogenesis related proteins was examined by immunoblotting. The intracellular cyclic AMP amount was evaluated by the cAMP Glo assay kit. The activity of phosphodiesterase 4B (PDE4B) was investigated by CREB reporter assay with overexpressed PDE4B or not. ResultsWe demonstrated that a low dose of IMQ could trigger melanogenesis in B16F10 cells. IMQ induced microphthalmia-associated transcription factor (MITF) nuclear translocation, upregulated the expression of melanogenesis-related proteins, increased tyrosinase (TYR) activity, and led to pigmentation in B16F10 cells. Next, we found that IMQ-induced melanogenesis was activated by excessive intracellular cAMP accumulation, which was regulated through IMQ-mediated PDE4B inhibition. Finally, IMQ-induced ROS production was found to be involved in melanogenesis by its control of PDE4B activity. ConclusionsLow dose of IMQ could activate melanogenesis through the ROS/PDE4B/PKA pathway in melanoma cells.

Design, Synthesis, and Structure-Activity Relationship Studies of Novel GPR88 Agonists (4-Substituted-phenyl)acetamides Based on the Reversed Amide Scaffold.

The development of synthetic agonists for the orphan receptor GPR88 has recently attracted significant interest, given the promise of GPR88 as a novel drug target for psychiatric and neurodegenerative disorders. Examination of structure-activity relationships of two known agonist scaffolds 2-PCCA and 2-AMPP, as well as the recently resolved cryo-EM structure of 2-PCCA-bound GPR88, led to the design of a new scaffold based on the "reversed amide" strategy of 2-AMPP. A series of novel (4-substituted-phenyl)acetamides were synthesized and assessed in cAMP accumulation assays as GPR88 agonists, which led to the discovery of several compounds with better or comparable potencies to 2-AMPP. Computational docking studies suggest that these novel GPR88 agonists bind to the same allosteric site of GPR88 that 2-PCCA occupies. Collectively, our findings provide structural insight and SAR requirement at the allosteric site of GPR88 and a new scaffold for further development of GPR88 allosteric agonists.

Potent MOR Agonists from 2′-Hydroxy-5,9-dimethyl-N-phenethyl Substituted-6,7-benzomorphans and from C8-Hydroxy, Methylene and Methyl Derivatives of N-Phenethylnormetazocine

(−)-5,9-Dimethyl-6,7-benzomorphan (normetazocine) derivatives with a para-OH or ortho-F substituent in the aromatic ring of the N-phenethyl moiety were synthesized and found to have subnanomolar potency at MOR, and both were fully efficacious in vitro. These new compounds, (1R,5R,9R)-6,11-dimethyl-3-(2-fluorophenethyl)-1,2,3,4,5,6-hexahydro-2,6-methanobenzo[d]azocin-8-ol and (1R,5R,9R)-6,11-dimethyl-3-(4-hydroxyphenethyl)-1,2,3,4,5,6-hexahydro-2,6-methanobenzo[d]azocin-8-ol, were more potent than the unsubstituted compound N-phenethylnormetazocine and about 30 or 40 times more potent than morphine, respectively. A variety of substituents in the ortho, meta, or para position in the aromatic ring of the N-phenethyl moiety were synthesized, 25 of these compounds, and found to have varying effects on potency and efficacy as determined by the forskolin-induced cAMP accumulation assay. The N-phenethyl moiety was also modified by increasing chain length to form a N-phenylpropyl side chain with and without a para-nitro moiety, and by an N-cinnamyl side chain. Also, an indole ethylamine normetazocine was synthesized to replace the N-phenethylamine side chain in normetazocine. The phenylpropylamine, propenylamine (cinnamyl) and the para-nitropropylamine had little or no MOR potency. The indole-ethylamine on the normetazocine nucleus, however, had moderate potency (MOR EC50 = 12 nM), and was fully efficacious (%Emax = 102%) in the cAMP assay. Retention of the N-phenethyl moiety and the addition of alkyl and alkenyl moieties on C8 in (−)-N-phenethylnormetazocine gave a C8-methylene derivative that had subnanomolar potency at MOR and a C8-methyl analog that had nanomolar potency. Five C8-substituted compounds were synthesized.

GLP-1 RAs and SGLT2i: two antidiabetic agents associated with immune and inflammation modulatory properties through the common AMPK pathway.

Immune cells and other cells respond to nutrient deprivation by the classic catabolic pathway of AMPK (Adenosine monophosphate kinase). This kinase is a pivotal regulator of glucose and fatty acids metabolism, although current evidence highlights its role in immune regulation. Indeed AMPK, through activation of Foxo1 (Forkhead box O1) and Foxo3 (Forkhead box O3), can regulate FOXP3, the key gene for differentiation and homeostasis of Tregs (T regulators lymphocytes). The relevance of Tregs in the onset of T1D (Type 1 diabetes) is well-known, while their role in the pathogenesis of T2D (Type 2 diabetes) is not fully understood yet. However, several studies seem to indicate that Tregs may oppose the progression of diabetic complications by mitigating insulin resistance, atherosclerosis, and damage to target organs (as in kidney disease). Hence, AMPK and AMPK-activating agents may play a role in the regulation of the immune system. The connection between metformin and AMPK is historically known; however, this link and the possible related immune effects are less studied about SGLT2i (Sodium-glucose co-transport 2 inhibitors) and GLP1-RAs (Glucagon-like peptide-1 receptor agonists). Actual evidence shows that the negative caloric balance, induced by SGLT2i, can activate AMPK. Conversely and surprisingly, an anabolizing agent like GLP-1RAs can also upregulate this kinase through cAMP (Cyclic adenosine monophosphate) accumulation. Therefore, both these drugs can likely lead to the activation of the AMPK pathway and consequential proliferation of Tregs. These observations seem to confirm not only the metabolic but also the immunoregulatory effects of these new antidiabetic agents.

Crustacean cardioactive peptide signaling system in the gastropod mollusk Pacific abalone

Crustacean cardioactive peptide (CCAP) signaling systems have been characterized in a diverse range of protostomes, representatively in arthropods. The cyclic CX5C-type CCAP regulates various biological activities through CCAP receptors (CCAPRs), which are orthologous to neuropeptide S receptors (NPSRs) in deuterostomes. However, the CCAPRs of the lophotrochozoa remain poorly characterized; therefore, the relationship between the CCAP, NPS, and CX4C-type oxytocin/vasopressin (OT/VP) signaling systems is unclear. In this study, we identified a CCAP precursor and two CCAPR isoforms in the Pacific abalone (Haliotis discus hannai; Hdh). The Hdh-CCAP precursor was found to harbor three CX5C-type and one CX4C-type CCAPs. The Hdh-CCAPRs displayed homology with protostome CCAPRs and deuterostome NPSRs, having characteristics of the rhodopsin-type G protein-coupled receptors. Phylogenetic analysis showed that lophotrochozoan CCAPRs, including Hdh-CCAPRs, form a monophyletic group distinct from arthropod CCAPRs. Reporter assays demonstrated that all examined Hdh-CCAPs and insect CCAP-induced intracellular Ca2+ mobilization and cAMP accumulation in Hdh-CCAPR-expressing HEK293 cells, whereas none of the CCAP peptides inhibited the forskolin-stimulated cAMP signaling pathway even at micromolar concentrations. In silico ligand-receptor docking models showed that the N-terminal FCN motifs of Hdh-CCAPs are deeply inserted inside the binding pocket of Hdh-CCAPR, forming extensive hydrophobic interactions. In mature Pacific abalone, the transcripts for Hdh-CCAP precursor and Hdh-CCAPR were highly expressed in the neural ganglia compared to the peripheral tissues. Collectively, this study characterized the first CCAP signaling system linked to both Ca2+/PKC and cAMP/PKA signal transduction pathways in gastropod mollusks and gives insights into the evolutional origins of deuterostomian NPS and OT/VP signaling systems.

Abstract 16052: Non-Canonical Mechanisms of BARKct-Mediated GRK2 Inhibition in Heart Failure Rescue

Introduction: Significant GRK2 upregulation during HF results in desensitization of βARs. βARKct is a peptide inhibitor of GRK2 comprised of the last carboxyl terminal region of GRK2, and prevents GRK2 binding and βAR desensitization. βARKct overexpression attenuates HF and improves outcomes in animal models. Following oxidative stress, MAPKs phosphorylate the Ser670 (S670) residue of GRK2, which induces GRK2 binding to Hsp90 and localization to mitochondria, initiating pro-death pathways. As S670 is also found in βARKct, it may prevent endogenous GRK2 accumulation in the mitochondria. Hypothesis: βARKct-mediated cardioprotection in HF is due primarily to mitochondrial GRK2 blockade by βARKct. Methods: We generated cardiac-specific mutant βARKct-S670A transgenic mice harboring a Ser-to-Ala mutation at the S670 residue to prevent Hsp90 binding and allow for endogenous GRK2 to continue to translocate to the mitochondria upon ischemic injury, while retaining βARKct in the cytosol to act on βAR signaling pathways. Additionally, we have generated AAV vectors to deliver WT βARKct, βARKct-S670A, and phosphomimetic βARKct-S670D peptides in mice. We performed in vivo hemodynamic analysis to assess cardiac function in βARKct and βARKct-S670A transgenic mice. Additionally, intracellular cAMP levels in AC16 cardiomyocytes transfected with βARKct or βARKct-S670A, and βARKct-S670D plasmids were quantified in response to increasing doses of isoproterenol. Results: Hemodynamic analysis of the βARKct-S670A mice has demonstrated increased baseline contractility in βARKct-S670A mice compared to NLC mice, indicating comparable cardioprotective effects to βARKct mice lacking the point mutation, which are mediated by canonical pathways. In vitro cAMP quantification revealed AC16s transfected with βARKct, βARKct-S670A, and βARKct-S670D plasmids increased cAMP accumulation in response to increasing doses of isoproterenol compared to control. Conclusions: Studies characterizing the mitochondrial pathways involved in HF rescue following ischemic injury are ongoing. These findings will identify new mechanistic information of GRK2 inhibition for HF, and elucidate a new method for attenuating mitochondrial dysfunction during HF.

Dynamic regulation of pancreatic β cell function and gene expression by the SND1 coregulator in vitro

ABSTRACT The pancreatic β cell synthesizes, packages, and secretes insulin in response to glucose-stimulation to maintain blood glucose homeostasis. Under diabetic conditions, a subset of β cells fail and lose expression of key transcription factors (TFs) required for insulin secretion. Among these TFs is Pancreatic and duodenal homeobox 1 (PDX1), which recruits a unique subset of transcriptional coregulators to modulate its activity. Here we describe a novel interacting partner of PDX1, the Staphylococcal Nuclease and Tudor domain-containing protein (SND1), which has been shown to facilitate protein-protein interactions and transcriptional control through diverse mechanisms in a variety of tissues. PDX1:SND1 interactions were confirmed in rodent β cell lines, mouse islets, and human islets. Utilizing CRISPR-Cas9 gene editing technology, we deleted Snd1 from the mouse β cell lines, which revealed numerous differentially expressed genes linked to insulin secretion and cell proliferation, including limited expression of Glp1r. We observed Snd1 deficient β cell lines had reduced cell expansion rates, GLP1R protein levels, and limited cAMP accumulation under stimulatory conditions, and further show that acute ablation of Snd1 impaired insulin secretion in rodent and human β cell lines. Lastly, we discovered that PDX1:SND1 interactions were profoundly reduced in human β cells from donors with type 2 diabetes (T2D). These observations suggest the PDX1:SND1 complex formation is critical for controlling a subset of genes important for β cell function and is targeted in diabetes pathogenesis.

SAT030 HM15912, A Novel Long-Acting GLP-2 Analog, Improves Intestinal Growth And Absorption Capacity In Rat Model Of Short Bowel Syndrome

Abstract Disclosure: J. Choi: Employee; Self; Hanmi Pharm. Co., Ltd. C. Park: Employee; Self; Hanmi Pharm. Co., Ltd. E. Park: Employee; Self; Hanmi Pharm. Co., Ltd. H. Kwon: Employee; Self; Hanmi Pharm. Co., Ltd. S. Bae: Employee; Self; Hanmi Pharm. Co., Ltd. D. Kim: Employee; Self; Hanmi Pharm. Co., Ltd. S. Lee: Employee; Self; Hanmi Pharm. Co., Ltd. I. Choi: Employee; Self; Hanmi Pharm. Co., Ltd. Short bowel syndrome (SBS) with intestinal failure requires partial or total parenteral nutrition (PN) to maintain health and growth. However, long-term use of PN may lead to life-threatening complications. Although teduglutide was firstly approved, the widespread use of it is still limited due to insufficient efficacy and leading to a significant burden for patients by daily administration. Hence, there is a medical unmet needs for more effective and longer lasting GLP-2 analog drugs. Here, we investigated the potential therapeutic effect of HM15912, which is currently under clinical development for once a month use, in small bowel resected rat model of SBS. To evaluate in vitro activity of HM15912, cAMP accumulation in CHO cells overexpressed human GLP-2 receptor (GLP-2R) was measured, and HM15912 was potently activated GLP-2R with a full agonistic activity as native human GLP-2 (EC50= 0.327 vs 0.173 nM, relative activity= 52.5%). Next, to demonstrate that GLP-2R stimulation by HM15912 results in the production and release of insulin-like growth factor-1 (IGF-1) as a known mechanism of GLP-2, HM15912 was treated to mice primary intestinal subepithelial myofibroblasts. mRNA transcription and protein secretion levels of IGF-1 was dose-dependently increased by treatment of HM15912 (p &amp;lt; 0.05 and p &amp;lt; 0.001, respectively). Longer-lasting property was evaluated in rats after single administration, and HM15912 exhibited 70-fold extended elimination half-life (42.4 h) compared to teduglutide (0.6 h). Based on this prolonged mode of action with the potent in vitro activity, intestinotrophic effect of HM15912 was investigated in 80% of small intestine (SI) resected jejuno-ileal anastomosis model rats. The SBS rats given HM15912 every week for 2 weeks significantly increased wet weight of jejunum compared to resection vehicle or b.i.d treatment of teduglutide. This result was well-correlated with histological analysis such as villus height, crypt depth, and mucosal area. In addition, HM15912 treated group was associated with a significant increase in absorption capacity of SI such as plasma D-xylose concentrations. Furthermore, the mice given HM15912 with various administration intervals for 2 weeks significantly increased SI weight compared to b.i.d treatment of teduglutide (35% over vehicle). In Q2D, Q4D and Q1W dosing intervals of HM15912, SI weights were dose-dependently increased 66∼112% (p &amp;lt; 0.001), 91∼103% (p &amp;lt; 0.001) and 55∼74% over vehicle (p &amp;lt; 0.05∼0.001), respectively. The results supported that small bowel hypertrophic effect of HM15912 are well-correlated with functional improvement of SI in short bowel condition, and superior efficacy to teduglutide was still observed even after once weekly administration in mice. Therefore, HM15912 could be a novel therapeutic option for SBS by providing remarkable small bowel tropic effect with extended administration interval. Presentation: Saturday, June 17, 2023