Selective Small Molecule Agonist Research Articles

Selective ligand recognition and activation of somatostatin receptors SSTR1 and SSTR3

Somatostatin receptors (SSTRs) exert critical biological functions such as negatively regulating hormone release and cell proliferation, making them popular targets for developing therapeutics to treat endocrine disorders, especially neuroendocrine tumors. Although several panagonists mimicking the endogenous ligand somatostatin are available, the development of more effective and safer somatostatinergic therapies is limited due to a lack of molecular understanding of the ligand recognition and regulation of divergent SSTR subtypes. Here, we report four cryoelectron microscopy structures of Gi-coupled SSTR1 and SSTR3 activated by distinct agonists, including the FDA-approved panagonist pasireotide as well as their selective small molecule agonists L-797591 and L-796778. Our structures reveal a conserved recognition pattern of pasireotide in SSTRs attributed to the binding with a conserved extended binding pocket, distinct from SST14, octreotide, and lanreotide. Together with mutagenesis analyses, our structures further reveal the dynamic feature of ligand binding pockets in SSTR1 and SSTR3 to accommodate divergent agonists, the key determinants of ligand selectivity lying across the orthosteric pocket of different SSTR subtypes, as well as the molecular mechanism underlying diversity and conservation of receptor activation. Our work provides a framework for rational design of subtype-selective SSTR ligands and may facilitate drug development efforts targeting SSTRs with improved therapeutic efficacy and reduced side effects.

From Biological Activity to Stereoselectivity: A Portrait of Molecular and Mechanistic Profiles of the Therapeutic Potential of G-1 and LNS8801 as GPER-1 Activator in the Treatment of Waldenström’s Macroglobulinemia

Therapeutic activation of G protein-coupled estrogen receptor 1 (GPER-1) shows promise for treating Waldenström's Macroglobulinemia (WM). Recently, the selective small-molecule agonist G-1 or its enantiomer LNS8801 were reported as potent GPER-1 pharmacological activators, however, the molecular events surrounding their chirality towards GPER-1 is still unexplored. This study aimed to explore the molecular events surrounding the chirality of the selective small-molecule agonists G-1 and LNS8801 towards GPER-1. Molecular docking and dynamics simulations revealed strong binding affinities and key amino acid residues involved in the activation process. The results revealed favourable binding affinities of -29.87kcal/mol, and -28.09kcal/mol for G-1 and LNS8801 towards GPER-1, respectively. Per-residue energy decomposition and time-dependent analysis proved that Arg253 and Arg254 are key amino acid residues in binding these activators towards GPER-1. The activators affected stability, flexibility, and structure of GPER-1. G-1 exhibited greater rigidity when bound to GPER-1 compared to LNS880. This study's findings illuminate chirality and the potential for optimizing the enantiomer ratio to enhance inhibitory effects. These results could also reveal the molecular specifics and binding mechanism of the G-1 and its enantiomer LNS8801 with GPER-1, laying the basis for the rational design of enhanced chiral molecules against WM.

An inverse agonist of orphan receptor GPR61 acts by a G protein-competitive allosteric mechanism

GPR61 is an orphan GPCR related to biogenic amine receptors. Its association with phenotypes relating to appetite makes it of interest as a druggable target to treat disorders of metabolism and body weight, such as obesity and cachexia. To date, the lack of structural information or a known biological ligand or tool compound has hindered comprehensive efforts to study GPR61 structure and function. Here, we report a structural characterization of GPR61, in both its active-like complex with heterotrimeric G protein and in its inactive state. Moreover, we report the discovery of a potent and selective small-molecule inverse agonist against GPR61 and structural elucidation of its allosteric binding site and mode of action. These findings offer mechanistic insights into an orphan GPCR while providing both a structural framework and tool compound to support further studies of GPR61 function and modulation.

Targeting EP2 Receptor for Drug Discovery: Strengths, Weaknesses, Opportunities, and Threats (SWOT) Analysis.

Cyclooxygenase-1 and -2 (COX1 and COX2) derived endogenous ligand prostaglandin-E2 (PGE2) triggers several physiological and pathological conditions. It mediates signaling through four G-protein coupled receptors, EP1, EP2, EP3, and EP4. Among these, EP2 is expressed throughout the body including the brain and uterus. The functional role of EP2 has been extensively studied using EP2 gene knockout mice, cellular models, and selective small molecule agonists and antagonists for this receptor. The efficacy data from in vitro and in vivo animal models indicate that EP2 receptor is a major proinflammatory mediator with deleterious functions in a variety of diseases suggesting a path forward for EP2 inhibitors as the next generation of selective anti-inflammatory and antiproliferative agents. Interestingly in certain diseases, EP2 action is beneficial; therefore, EP2 agonists seem to be clinically useful. Here, we highlight the strengths, weaknesses, opportunities, and potential threats (SWOT analysis) for targeting EP2 receptor for therapeutic development for a variety of unmet clinical needs.

Abstract P2047: Identification Of Selective Small-molecule ERBB4 Agonists With Cardioprotective Effects

Introduction: Morbidity and mortality of heart failure remain high, mandating new therapeutic approaches. The neuregulin-1 (NRG1)/ERBB4 axis is cardioprotective and an attractive target for treatment. Clinical trials with recombinant NRG1 are ongoing, but require intravenous administration, limiting applicability and efficacy. Purpose: To develop selective small-molecule ERBB4 agonists with cardioprotective effects. Methods: A high-throughput screening of 10,240 compounds (cpds) was performed on a ERBB4/ERBB4 dimerization assay. Hit cpds were co-administered with NRG1 or fluorescently labeled NRG1 to determine competitive binding. Selectivity, receptor phosphorylation, toxicity and metabolic stability were determined using Luminex RTK phosphoprotein, ERBB2/ERBB3 dimerization and adenylate kinase assays or LC-MS/MS. Apoptotic and hypertrophic effects of cpds (4-32μM) on cultured cardiomyocytes were studied after exposure to 100μM H 2 O 2 and 100nM angiotensin II (AngII). Antifibrotic effects (4-32μM) were studied on TGF-β-induced collagen synthesis in cultured human fibroblasts, and in mice (n=9-10/group) treated with AngII (1000 ng/kg/min) or cpd (83 μg/kg/h) using osmotic pumps. Results: We identified 8 similar pyrimidine derivatives inducing ERBB4/ERBB4 dimerization (Emax 9-33% relative to NRG1, EC50 6E-6 to 2E-7M). Competition assays indicated allosteric receptor binding and potentiation of NRG1-induced ERBB4 receptor dimerization, up to 2.7 fold (P<0.0001). Six were non-toxic and induced ERBB4 phosphorylation, but ERBB1, ERBB2 or ERBB3 phosphorylation remained unaffected and cpds did not induce ERBB2/ERBB3 dimerization, showing ERBB4 selectivity. Cpds showed a t1/2 of 170min in human liver microsomes. Cpds attenuated hypertrophic and apoptotic effects of AngII or H 2 O 2 (P<0.05), and decreased collagen upregulation in vitro (P<0.05). In vivo , a selected cpd attenuated AngII-induced myocardial interstitial fibrosis (-76±26%, P<0.01), and cardiac Col1a1, Col3a1 (-64±22%; -71±25%, P<0.01) and Nppa (-77±22%, P<0.01) mRNA expression. Conclusion: We identified selective novel pyrimidine derivative small-molecule ERBB4 agonists with cardioprotective effects in vitro and in vivo .

Pharmacological Melanocortin 5 Receptor Activation Attenuates Glomerular Injury and Proteinuria in Rats With Puromycin Aminonucleoside Nephrosis.

Clinical evidence indicates that the melanocortin peptide ACTH is effective in inducing remission of nephrotic glomerulopathies like minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), including those resistant to steroids. This suggests that a steroid-independent melancortinergic mechanism may contribute. However, the type of melanocortin receptor (MCR) that conveys this beneficial effect as well as the underlying mechanisms remain controversial. Burgeoning evidence suggests that MC5R is expressed in glomeruli and may be involved in glomerular pathobiology. This study aims to test the effectiveness of a novel highly selective MC5R agonist (MC5R-A) in puromycin aminonucleoside (PAN) nephrosis. Upon PAN injury, rats developed evident proteinuria on day 5, denoting an established nephrotic glomerulopathy. Following vehicle treatment, proteinuria continued to persist on day 14 with prominent histologic signs of podocytopathy, marked by ultrastructural glomerular lesions, including extensive podocyte foot process effacement. Concomitantly, there was loss of podocyte homeostatic markers, such as synaptopodin and podocin, and de novo expression of the podocyte injury marker desmin. Treatment with MC5R-A attenuated urine protein excretion and mitigated the loss of podocyte marker proteins, resulting in improved podocyte ultrastructural changes. In vitro in cultured podocytes, MC5R-A prevented the PAN-induced disruption of actin cytoskeleton integrity and apoptosis. MC5R-A treatment in PAN-injured podocytes also reinstated inhibitory phosphorylation and thus averted hyperactivity of GSK3β, a convergent point of multiple podocytopathic pathways. Collectively, pharmacologic activation of MC5R by using the highly selective small-molecule agonist is likely a promising therapeutic strategy to improve proteinuria and glomerular injury in protenuric nephropathies.

Delayed Treatment with a Novel Highly Selective Small-Molecule Agonist of MC5R Attenuates Podocyte Injury and Proteinuria in Puromycin Aminonucleoside Nephrosis

Delayed Treatment with a Novel Highly Selective Small-Molecule Agonist of MC5R Attenuates Podocyte Injury and Proteinuria in Puromycin Aminonucleoside Nephrosis

RORγt agonist synergizes with CTLA-4 antibody to inhibit tumor growth through inhibition of Treg cells via TGF-β signaling in cancer

To date, the overall response rate to checkpoint blockade remains unsatisfactory, partially due to the limited understanding of the tumor immune microenvironment. The retinoic acid-related orphan receptor γt (RORγt) is the key transcription factor of T helper cell 17 (Th17) cells and plays an essential role in tumor immunity. In this study, we used JG-1, a potent and selective small-molecule RORγt agonist to evaluate the therapeutic potential and mechanism of action of targeting RORγt in tumor immunity. JG-1 promotes Th17 cells differentiation and inhibition of regulatory T (Treg) cells differentiation. JG-1 demonstrates robust tumor growth inhibition in multiple syngeneic models and shows a synergic effect with the Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) antibody. In tumors, JG-1 not only promotes Th17 cells differentiation and increases C-C Motif Chemokine Receptor 6 (CCR6)- Chemokine (C-C motif) ligand 20 (CCL20) expression, but also inhibits both the expression of transforming growth factor-β1 (TGF-β1) and the differentiation and infiltration of Treg cells. In summary, JG-1 is a lead compound showing a potent activity in vitro and robust tumor growth inhibition in vivo with synergetic effects with anti-CTLA-4.

Abstract 1280: New generation of STING agonists: Development and characterization of a novel series of systemic immunomodulators with improved potency

Abstract Background. Stimulator of interferon genes, known as STING, is an intracellular sensor of nucleic acids and one of key regulators in activating the innate immune response. Employing synthetic STING agonists has been shown to promote immune-mediated antitumor response in preclinical animal models. Ryvu is developing small-molecule STING agonists suitable for systemic administration. Herein we present unpublished results from characterization of the new generation of our agonist series with significantly improved potency on human immune cells. Methods. Binding to recombinant STING protein was examined using Fluorescence Thermal Shift and Fluorescence Polarisation and was confirmed by X-ray crystallography. Primary screen was performed in THP-1 Dual reporter cells and selectivity was confirmed in THP-1 reporter cells with knocked out STING or expressing varying STING variants. T cell viability and proliferation was assessed by flow cytometry using activated, human T cells exposed to STING agonists. STING pathway activation pattern in cells treated with Ryvu's molecules was confirmed using Western blot analysis. BALB/c mice were injected with compounds and the levels of cytokine release were measured in the plasma. Mice were inoculated with CT26 or EMT6 tumor cells and the compound was administered intravenously followed by the regular monitoring of tumor growth. Results. New generation Ryvu STING agonists are strong binders of human STING protein. Ryvu's compounds show high cellular potency inducing cytokine production in human immune cells at low nM range. Moreover, high activity of developed agonists is maintained irrespective of the natural human STING variant as seen in THP-1 reporter cells as well as in human primary immune cells. High cellular potency of developed compounds also translates into efficacy observed in vivo, where systemic intravenous administration leads to significant tumor growth inhibition and complete tumor regressions in mouse syngeneic models. Conclusion. Ryvu has developed a new generation of potent, direct and selective small-molecule STING agonists. The compounds are characterized by drug-like properties and high in vitro potency on par or outperforming known references. Ryvu agonists are suitable for systemic administration and allow to achieve excellent antitumor efficacy. Taken together, the promising results suggest that the developed series holds high potential for improving immunotherapy in cancer patients. Citation Format: Maciej Krzysztof Rogacki, Stefan Chmielewski, Jolanta Mazurek, Magdalena Zawadzka, Katarzyna Wnuk-Lipińska, Kamil Kuś, Katarzyna Wójcik-Jaszczyńska, Aleksandra Poczkaj, Łukasz Dudek, Wojciech Schonemann, Urszula Głowniak-Kwitek, Marcin Leś, Marek Wronowski, Tushar Mahajan, Urszula Kulesza, Magdalena Zastawna, David Synak, Karol Zuchowicz, Karolina Gluza, Katarzyna Banaszak, Karolina Wiatrowska, Izabela Strojny, Mirosława Gładysz, Justyna Jabłońska, Ewelina Gabor-Worwa, Monika Dobrzańska, Raghuram Tangirala, Peter Littlewood, Krzysztof Brzózka. New generation of STING agonists: Development and characterization of a novel series of systemic immunomodulators with improved potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1280.

Phase 1 trial of a novel, first-in-class G protein-coupled estrogen receptor (GPER) agonist, LNS8801, in patients with advanced or recurrent treatment-refractory solid malignancies.

3084 Background: The G protein-coupled estrogen receptor (GPER) is a broadly expressed G protein-coupled receptor that is tumor suppressive. LNS8801 is an oral, highly selective small molecule agonist of GPER. GPER activation results in c-Myc depletion, inhibition of tumor proliferation, and enhancement of tumor immune recognition. Preclinically, LNS8801 demonstrates potent single-agent and combinatorial anti-cancer activity and can overcome established resistance to standard-of-care anti-cancer therapies including immune checkpoint inhibitors. Methods: The primary objective of this phase 1/1B first-in-human, open-label, multicenter study (NCT04130516) was to determine the safety and tolerability and recommended phase 2 dose (RP2D) of LNS8801 in patients (pts) with locally advanced or metastatic solid tumor malignancies, both as monotherapy and in combination with the anti-PD-1 antibody, pembrolizumab. Dose levels were escalated in a 3+3 fashion and included 10, 40 and 125 mg dosed 3/7, 125 mg daily, and 125 and 250 mg twice daily. Dose limiting toxicity (DLT) was defined via NCI CTCAE v5.0 during the first 21 days of treatment. An increase in prolactin over the initial 12 hrs of dosing was measured to assess systemic GPER signaling. Tumor c-Myc expression was measured as a surrogate of treatment-related biologic response. Radiographic response (RECIST v1.1) was evaluated every 8 weeks until progression. Results: 33 pts (19 M/14 F) with median age 58.8 y and 4 (1-9) prior therapies enrolled. Median duration of treatment was 66 d (1–367+). With monotherapy (n = 28), no DLTs, treatment-related SAEs, or treatment-related study discontinuations were observed up through the maximum administered dose (250 mg bid). Possibly related AEs were grade 1 or 2 and did not correlate with dose level. Exposure was above that predicted for efficacy and t1/2 was ̃10 hr at all doses. Of 26 evaluable monotherapy pts, 8 (27%) experienced stable disease (SD) for up to a year. All SD pts had a prolactin response. Among tumors expressing GPER, c-Myc depletion was observed in 100% (5/5) of paired pre and on-treatment biopsies. In the combination cohort (n = 5), 2/2 evaluable pts demonstrated net tumor reductions on initial f/up scans, including one RECIST partial response. Based on PK/PD data, 125 mg daily has provisionally been identified as the monotherapy and combination RP2D. Conclusions: LNS8801 is well tolerated and demonstrates signals of anti-tumor activity when administered both as monotherapy and in combination with pembrolizumab. Confirmation of RP2Ds and updated efficacy data will be presented in June. A phase 2A expansion study to evaluate these RP2Ds in clinical settings of high unmet need is now in development. Clinical trial information: NCT04130516.

Development of CVN424: A Selective and Novel GPR6 Inverse Agonist Effective in Models of Parkinson Disease.

GPR6 is an orphan G-protein-coupled receptor that has enriched expression in the striatopallidal, indirect pathway and medium spiny neurons of the striatum. This pathway is greatly impacted by the loss of the nigro-striatal dopaminergic neurons in Parkinson disease, and modulating this neurocircuitry can be therapeutically beneficial. In this study, we describe the in vitro and in vivo pharmacological characterization of (R)-1-(2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-3-((tetrahydrofuran-3-yl)amino)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)ethan-1-one (CVN424), a highly potent and selective small-molecule inverse agonist for GPR6 that is currently undergoing clinical evaluation. CVN424 is brain-penetrant and shows dose-dependent receptor occupancy that attained brain 50% of receptor occupancy at plasma concentrations of 6.0 and 7.4 ng/ml in mice and rats, respectively. Oral administration of CVN424 dose-dependently increases locomotor activity and reverses haloperidol-induced catalepsy. Furthermore, CVN424 restored mobility in bilateral 6-hydroxydopamine lesion model of Parkinson disease. The presence and localization of GPR6 in medium spiny neurons of striatum postmortem samples from both nondemented control and patients with Parkinson disease were confirmed at the level of both RNA (using Nuclear Enriched Transcript Sort sequencing) and protein. This body of work demonstrates that CVN424 is a potent, orally active, and brain-penetrant GPR6 inverse agonist that is effective in preclinical models and is a potential therapeutic for improving motor function in patients with Parkinson disease. SIGNIFICANCE STATEMENT: CVN424 represents a nondopaminergic novel drug for potential use in patients with Parkinson disease.

Toll-Like Receptor 8 Agonist GS-9688 Induces Sustained Efficacy in the Woodchuck Model of Chronic Hepatitis B.

Background and AimsGS‐9688 (selgantolimod) is an oral selective small molecule agonist of toll‐like receptor 8 in clinical development for the treatment of chronic hepatitis B. In this study, we evaluated the antiviral efficacy of GS‐9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus.Approach and ResultsWHV‐infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS‐9688, or 3 mg/kg GS‐9688. Vehicle and 1 mg/kg GS‐9688 had no antiviral effect, whereas 3 mg/kg GS‐9688 induced a >5 log10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS‐9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti‐WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS‐9688 was confirmed in a second woodchuck study. The antiviral response to GS‐9688 did not correlate with systemic GS‐9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells and various other immune cell subsets may play a role in the antiviral response to GS‐9688.ConclusionsFinite, short‐duration treatment with a clinically relevant dose of GS‐9688 is well tolerated and can induce a sustained antiviral response in WHV‐infected woodchucks; the identification of a baseline intrahepatic transcriptional signature associated with response to GS‐9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect.

Agonism of adiponectin Receptor Prevents 7‐ketocholesterol‐induced Endothelial Inflammation through Suppressing Endoplasmic Reticulum and Oxidative Stresses

Endoplasmic reticulum (ER) stress is involved in endothelial inflammation and injury by various cardiovascular risk factors including oxysterols. AdipoRon is a selective small‐molecule agonist of adiponectin receptor, which has been shown to mimics many beneficial effects of adiponectin on the cardiovascular system including endothelial cells. However, the molecular mechanisms that mediate adipoRon‐induced endothelial protection are not well understood. Here, we studied whether adipoRon treatment interferes with the induction of ER stress to protect the endothelial cells from oxysterol‐induced inflammation and injury in vitro and to inhibit vascular inflammation from acute hypercholesterolemia in vivo. In cultured murine endothelial cells, adipoRon prevented 7‐ketocholesterol‐induced increases in endothelial cell death, inflammatory gene expression, monocyte adhesion, and Nlrp3 inflammasome activation. In parallel, adipoRon inhibited 7‐ketocholesterol‐induced ER stress and reactive oxygen species (ROS) production. In mice with acute hypercholesterolemia, adipoRon pre‐treatment could inhibit ROS production and ER stress in the coronary arterial wall without alterations in the plasma cholesterol concentration. Moreover, adipoRon reduced expression of ICAM1 in the coronary arterial intima and decreased numbers of CD45+ immune cells that adhere to or accumulate around the coronary arteries. Together, our results indicate that the cardiovascular protective action of adipoRon is associated with its inhibitory effects on ROS production and ER stress and consequent inflammatory responses in endothelial cells.Support or Funding InformationNational Institutes of Health (HL122769 and HL122937)

Selective Toll-like receptor 7 agonists with novel chromeno[3,4-d]imidazol-4(1H)-one and 2-(trifluoromethyl)quinoline/ quinazoline-4-amine scaffolds

Toll-like receptors (TLRs) are promising targets for treatment of viral infections, autoimmune diseases, and cancers. Here, two new series of selective small-molecule TLR7 agonists with novel scaffolds and good selectivity over TLR8 are described, some with potencies in the low micromolar range. 8-Hydroxy-1-isobutylchromeno[3,4-d]imidazol-4(1H)-one (26) from the first series was designed and synthesized on the basis of previously described TLR7 antagonist 2, and is shown to be a selective TLR7 agonist (EC50, 1.8 μM). The second series was based on 2-(trifluoromethyl)quinolin-4-amine and 2-(trifluoromethyl)quinazolin-4-amine scaffolds, which were defined according to our in-house ligand-based virtual screening protocol. Further synthesis of a focused library of analogs, biological evaluation, and docking studies provided systematic exploration of the structure−activity relationships, which indicate that a secondary or tertiary amine with smaller flexible alkyl substituents up to three carbon atoms in length, or bulkier rigid aliphatic rings is required at position 4 on 2-(trifluoromethyl)quinoline/quinazoline scaffold for potent TLR7 agonist activity. The influence of selected TLR7 agonists on cytokine production is also reported showing that N-cyclopropyl-2-(trifluoromethyl)quinazolin-4-amine (46) is able to induce increased levels of IL-6 and IL-8. These data demonstrate successful in-silico definition of novel TLR7 versus TLR8-selective compounds as promising chemical probes for further development of potent small-molecule immunomodulators.

Selective activation of Dopamine D3 receptors and norepinephrine transporter blockade enhances sustained attention

Catecholamine transmitters dopamine (DA) and norepinephrine (NE) regulate prefrontal cortical (PFC) circuit activity and PFC-mediated executive functions. Accordingly, pharmacological agents that influence catecholamine neurotransmission exert prominent effects on cognition. Many such agents are used clinically to treat attention disorders. For example, methylphenidate blocks DA and NE reuptake and is the leading choice for attention deficit hyperactivity disorder (ADHD) treatment. Recently, we have designed SK609 – a selective small molecule agonist of the DA D3 receptor (D3R). In this study, we further characterized SK609's ability to selectively inhibit the reuptake of NE by NE transporters (NET). Our results indicate SK609 selectively inhibits NET with a Ki value of ∼500 nM and behaves as a NET substrate. Systemic dosing of SK609 (4 mg/kg; i.p.) in naïve rats produced a 300% and 160% increase in NE and DA, respectively, in the PFC as measured by microdialysis. Based on these neurochemical results, SK609 was tested in a PFC-dependent, visually-guided sustained attention task in rats. SK609 improved performance in a dose-dependent manner with a classical inverted-U dose response function with a peak effect at 4 mg/kg. SK609's peak effect was blocked by a pre-treatment with either the D2/D3R antagonist raclopride (0.05 mg/kg; i.p) or the alpha-1 adrenergic receptor antagonist prazosin (0.25 mg/kg; i.p), confirming a role for both DA and NE in promoting sustained attention. Additionally, SK609 improved sustained attention more prominently among low-performing animals. Doses of SK609 (2, 4, and 8 mg/kg) associated with cognitive enhancement did not produce an increase in spontaneous locomotor activity, suggesting a lack of side effects mediated by DA transporter (DAT) activity. These results demonstrate that the novel catecholaminergic modulator SK609 has the potential to treat sustained attention deficits without affecting DAT activity, distinguishing it from amphetamines and methylphenidate.

Yoda1‐induced phosphorylation of the Akt and ERK1/2 does not require Piezo1 activation

Piezo1 is a mechanosensitive cation channel that is expressed by endothelial cells (ECs) and can be activated by fluid shear stress. It has been previously shown to be important for a number of shear‐induced EC responses, ranging from increased intracellular calcium to cell alignment. Most recently, Piezo1 has been implicated as a critical regulator of vascular tone and blood pressure. Yoda1, a selective small molecule agonist of Piezo1, has been shown to induce responses similar to those activated by fluid shear stress in ECs, such as calcium influx. Since shear stress‐induced intracellular calcium release is known to cause activation of Akt and ERK1/2, we sought to determine whether Yoda1 stimulation of EC can lead to similar responses and whether they are Piezo1‐mediated. In this study, we show that Yoda1 can robustly activate both the Akt and ERK1/2 signaling pathways in ECs. Additionally, we found that the ion channel antagonists, gadolinium and ruthenium red, but not the specific Piezo‐blocking peptide, GsMTx4, effectively blocks Yoda1‐induced Akt activation. Taken together, our results suggest that despite Yoda1 being a selective Piezo1 agonist, Yoda1‐induced activation of these two independent signaling pathways is not entirely dependent on Piezo1 activation.Support or Funding InformationThis work was supported by NIH MERIT Award R37 HL040696 to J.A.F.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Incretin-like effects of small molecule trace amine-associated receptor 1 agonists

ObjectiveType 2 diabetes and obesity are emerging pandemics in the 21st century creating worldwide urgency for the development of novel and safe therapies. We investigated trace amine-associated receptor 1 (TAAR1) as a novel target contributing to the control of glucose homeostasis and body weight.MethodsWe investigated the peripheral human tissue distribution of TAAR1 by immunohistochemistry and tested the effect of a small molecule TAAR1 agonist on insulin secretion in vitro using INS1E cells and human islets and on glucose tolerance in C57Bl6, and db/db mice. Body weight effects were investigated in obese DIO mice.ResultsTAAR1 activation by a selective small molecule agonist increased glucose-dependent insulin secretion in INS1E cells and human islets and elevated plasma PYY and GLP-1 levels in mice. In diabetic db/db mice, the TAAR1 agonist normalized glucose excursion during an oral glucose tolerance test. Sub-chronic treatment of diet-induced obese (DIO) mice with the TAAR1 agonist resulted in reduced food intake and body weight. Furthermore insulin sensitivity was improved and plasma triglyceride levels and liver triglyceride content were lower than in controls.ConclusionsWe have identified TAAR1 as a novel integrator of metabolic control, which acts on gastrointestinal and pancreatic islet hormone secretion. Thus TAAR1 qualifies as a novel and promising target for the treatment of type 2 diabetes and obesity.

GPR139, an Orphan Receptor Highly Enriched in the Habenula and Septum, Is Activated by the Essential Amino Acids L-Tryptophan and L-Phenylalanine.

GPR139 is an orphan G-protein-coupled receptor expressed in the central nervous system. To identify its physiologic ligand, we measured GPR139 receptor activity from recombinant cells after treatment with amino acids, orphan ligands, serum, and tissue extracts. GPR139 activity was measured using guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding, calcium mobilization, and extracellular signal-regulated kinases phosphorylation assays. Amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) activated GPR139, with EC50 values in the 30- to 300-μM range, consistent with the physiologic concentrations of L-Trp and L-Phe in tissues. Chromatography of rat brain, rat serum, and human serum extracts revealed two peaks of GPR139 activity, which corresponded to the elution peaks of L-Trp and L-Phe. With the purpose of identifying novel tools to study GPR139 function, a high-throughput screening campaign led to the identification of a selective small-molecule agonist [JNJ-63533054, (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl) benzamide]. The tritium-labeled JNJ-63533054 bound to cell membranes expressing GPR139 and could be specifically displaced by L-Trp and L-Phe. Sequence alignment revealed that GPR139 is highly conserved across species, and RNA sequencing studies of rat and human tissues indicated its exclusive expression in the brain and pituitary gland. Immunohistochemical analysis showed specific expression of the receptor in circumventricular regions of the habenula and septum in mice. Together, these findings suggest that L-Trp and L-Phe are candidate physiologic ligands for GPR139, and we hypothesize that this receptor may act as a sensor to detect dynamic changes of L-Trp and L-Phe in the brain.

G protein-coupled estrogen receptor protects from atherosclerosis.

Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity.

Discovery, design and synthesis of novel potent and selective sphingosine-1-phosphate 4 receptor (S1P4-R) agonists

High affinity and selective small molecule agonists of the S1P(4) receptor (S1P(4)-R) may have significant therapeutic utility in diverse disease areas including autoimmune diseases, viral infections and thrombocytopenia. A high-throughput screening (HTS) of the Molecular Libraries-Small Molecule Repository library identified 3-(2-(2,4-dichlorophenoxy)ethoxy)-6-methyl-2-nitropyridine as a moderately potent and selective S1P(4)-R hit agonist. Design, synthesis and systematic structure-activity relationships study of the HTS-derived hit led to the development of novel potent S1P(4)-R agonists exquisitely selective over the remaining S1P(1-3,5)-Rs family members. Remarkably, the molecules herein reported provide novel pharmacological tools to decipher the biological function and assess the therapeutic utility of the S1P(4)-R.