Potent Small-molecule Agonist Research Articles

Scaffold Hopping and Screening for Potent Small Molecule Agonists for GRP94: Implications to Alleviate ER Stress-Associated Pathogenesis.

Disparity in the activity of Endoplasmic reticulum (ER) leads to degenerative diseases, mainly associated with protein misfolding and aggregation leading to cellular dysfunction and damage, ultimately contributing to ER stress. ER stress activates the complex network of Unfolded Protein Response (UPR) signaling pathways mediated by transmembrane proteins IRE1, ATF6, and PERK. In addition to UPR, many ER chaperones have evolved to optimize the output of properly foldedsecretory and membrane proteins. Glucose-regulated protein 94 (GRP94), an ER chaperone of heat shock protein HSP90 family, directs protein folding through interaction with other components of the ER protein folding machinery and assists in ER-associated degradation (ERAD). Activation of GRP94 would increase the efficacy of protein folding machinery and regulate the UPR pathway toward homeostasis. The present study aims to screen for novel agonists for GRP94 based on Core hopping, pharmacophore hypothesis, 3D-QSAR, and virtual screening with small-molecule compound libraries in order to improve the efficiency of native protein folding by enhancing GRP94 chaperone activity, therefore to reduce protein misfolding and aggregation. In this study, we have employed the strategy of small molecule-dependent ER programming to enhance the chaperone activity of GRP94 through scaffold hopping-based screening approach to identify specific GRP94 agonists. New scaffolds generated by altering the cores of NECA, the known GRP94 agonist, were validated by employing pharmacophore hypothesis testing, 3D-QSAR modeling, and molecular dynamics simulations. This facilitated the identification of small molecules to improve the efficiency of native protein folding by enhancing GRP94 activity. High-throughput virtual screening of the selected pharmacophore hypothesis against Selleckchem and ZINC databases retrieved a total of 2,27,081 compounds. Further analysis on docking and ADMET properties revealed Epimedin A, Narcissoside, Eriocitrin 1,2,3,4,6-O-Pentagalloylglucose, Secoisolariciresinol diglucoside, ZINC92952357, ZINC67650204, and ZINC72457930 as potential lead molecules. The stability and interaction of these small molecules were far better than the known agonist, NECA indicating their efficacy in selectively alleviating ER stress-associated pathogenesis. These results substantiate the fact that small molecule-dependent ER reprogramming would activate the ER chaperones and therefore reduce the protein misfolding as well as aggregation associated with ER stress in order to restore cellular homeostasis.

Cardiovascular response to small-molecule APJ activation.

Heart failure (HF) remains a grievous illness with poor prognosis even with optimal care. The apelin receptor (APJ) counteracts the pressor effect of angiotensin II, attenuates ischemic injury, and has the potential to be a novel target to treat HF. Intravenous administration of apelin improves cardiac function acutely in patients with HF. However, its short half-life restricts its use to infusion therapy. To identify a longer acting APJ agonist, we conducted a medicinal chemistry campaign, leading to the discovery of potent small-molecule APJ agonists with comparable activity to apelin by mimicking the C-terminal portion of apelin-13. Acute infusion increased systolic function and reduced systemic vascular resistance in 2 rat models of impaired cardiac function. Similar results were obtained in an anesthetized but not a conscious canine HF model. Chronic oral dosing in a rat myocardial infarction model reduced myocardial collagen content and improved diastolic function to a similar extent as losartan, a RAS antagonist standard-of-care therapy, but lacked additivity with coadministration. Collectively, this work demonstrates the feasibility of developing clinical, viable, potent small-molecule agonists that mimic the endogenous APJ ligand with more favorable drug-like properties and highlights potential limitations for APJ agonism for this indication.

Discovery and SAR of aryl hydroxy pyrimidinones as potent small molecule agonists of the GPCR APJ

This article describes the discovery of aryl hydroxy pyrimidinones and the medicinal chemistry efforts to optimize this chemotype for potent APJ agonism. APJ is a G-protein coupled receptor whose natural agonist peptide, apelin, displays hemodynamic improvement in the cardiac function of heart failure patients. A high throughput screen was undertaken to identify small molecule hits that could be optimized to mimic the apelin in vitro response. A potent and low molecular weight aryl hydroxy pyrimidinone analog 30 was identified through optimization of an HTS hit and medicinal chemistry efforts to improve its properties.

Identification of potent pyrazole based APELIN receptor (APJ) agonists

The apelinergic system comprises the apelin receptor and its cognate apelin and elabela peptide ligands of various lengths. This system has become an increasingly attractive target for pulmonary and cardiometabolic diseases. Small molecule regulators of this receptor with good drug-like properties are needed. Recently, we discovered a novel pyrazole based small molecule agonist 8 of the apelin receptor (EC50 = 21.5 µM, Ki = 5.2 µM) through focused screening which was further optimized to initial lead 9 (EC50 = 0.800 µM, Ki = 1.3 µM). In our efforts to synthesize more potent agonists and to explore the structural features important for apelin receptor agonism, we carried out structural modifications at N1 of the pyrazole core as well as the amino acid side-chain of 9. Systematic modifications at these two positions provided potent small molecule agonists exhibiting EC50 values of <100 nM. Recruitment of β-arrestin as a measure of desensitization potential of select compounds was also investigated. Functional selectivity was a feature of several compounds with a bias towards calcium mobilization over β-arrestin recruitment. These compounds may be suitable as tools for in vivo studies of apelin receptor function.

Identification of Potent Pyrazole‐Based Biased Small Molecule APJ Receptor Agonists

The apelinergic system comprises the APJ receptor and its cognate apelin and ELABELA peptides of various lengths. This system has become an increasingly attractive target for cardiometabolic diseases. However, in vivo studies of this receptor are difficult because endogenous peptides are labile with short half‐lives and small molecule ligands are under reported. Recently, we discovered a novel pyrazole based small molecule agonist of APJ (EC50 = 21.5 μM, Ki = 5.2 μM) through focused screening of a compound library, which was further optimized to our initial lead (EC50 = 800 nM, Ki = 1.3 μM). In our efforts to synthesize more potent agonists and to explore the structural features important for APJ agonism, we carried out structural modifications at the pyrazole core as well as the amino acid side‐chain of our initial lead. Systematic modifications at these two positions provided potent small molecule agonists exhibiting EC50 values of ~100 nM using separate calcium mobilization and cAMP generation functional assays. Recruitment of β‐arrestin as a measure of desensitization potential of select compounds was also investigated. Functional selectivity was a feature of several compounds with a bias towards calcium mobilization over β‐arrestin recruitment. These compounds may be suitable for chronic in vivo studies of APJ and await additional characterization.Support or Funding InformationNIH Grants: 1R01HD079547‐01A1 and 1R01DK103625‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TLR1/2 Specific Small-Molecule Agonist Suppresses Leukemia Cancer Cell Growth by Stimulating Cytotoxic T Lymphocytes.

Toll‐like receptor 2 (TLR2) expressed on antigen presenting cells evokes a series of critical cytokines, which favor the development of tumor‐specific cytotoxic T lymphocytes (CTLs). Therefore, TLR2 represents an attractive cancer immunotherapeutic target. Here, a synthetic library of 14 000 compounds together with a series of newly developed compounds for NF‐κB activation using HEK‐Blue hTLR2 cells is initially screened. Following further screening in a variety of cells including HEK‐Blue hTLRs reporter cells, murine, and human macrophage cell lines, a potent small molecule agonist 23 (SMU‐Z1) is identified, which specifically activates TLR2 through its association with TLR1, with a EC50 of 4.88 ± 0.79 × 10−9 m. Toxicology studies, proinflammatory cytokines (e.g., TNF‐α, IL‐1β, IL‐6, and nitric oxide) and target‐protein based biophysical assays demonstrate the pharmacologically relevant characteristics of SMU‐Z1. In addition, SMU‐Z1 promotes murine splenocyte proliferation and upregulates the expression of CD8+ T cells, NK cells and DCs, which results in a significant antitumor effect in a murine leukemia model. Finally, the induced tumors in three out of seven mice disappear after administration of SMU‐Z1. Our studies thus identify a novel and potent TLR1/2 small molecule agonist, which displays promising immune adjuvant properties and antitumor immunity.

Development of time resolved fluorescence resonance energy transfer-based assay for FXR antagonist discovery

FXR (farnesoid X receptor, NRIH4), a nuclear receptor, plays a major role in the control of cholesterol metabolism. FXR ligands have been investigated in preclinical studies for targeted therapy against metabolic diseases, but have shown limitations. Therefore, there is a need for new agonist or antagonist ligands of FXR, both for potential clinical applications, as well as to further elucidate its biological functions. Here we describe the use of the X-ray crystal structure of FXR complexed with the potent small molecule agonist GW4064 to design and synthesize a novel fluorescent, high-affinity probe (DY246) for time resolved fluorescence resonance energy transfer (TR-FRET) assays. We then used the TR-FRET assay for high throughput screening of a library of over 5000 bioactive compounds. From this library, we identified 13 compounds that act as putative FXR transcriptional antagonists.

Integrin activation as an alternative treatment approach for inflammatory diseases

Regulation of immune responses is a complex process that involves many signaling molecules in their specific interactions and interplays. For instance, the leukocytic integrin CD11b/CD18 plays a crucial role in leukocyte infiltration, which is commonly found in most inflammatory diseases. The highly abundant integrin CD11b/CD18 is a heterodimer of the αm (CD11b) and β2 (CD18) subunits. CD11b/CD18 is located in the cell surface of circulating leukocytes and can undergo active conformational change to mediate leukocyte adhesion, migration and accumulation at the sites of inflammation1. Current approaches for prevention of leukocyte infiltration focus on inhibiting the adhesion of leukocytic integrins to their respective ligands. For example, the specific integrin antibody M1/70 mAb blocks the binding of integrins to ligands found on the vascular wall, thereby reducing the infiltration of leukocytes into the inflamed tissues (Figure 1)2, 3. Despite the intensive research on identifying potent inhibitors for CD11b/CD18, such inhibitors had limited success in treating inflammatory diseases in humans. This may be because of the large intracellular pool of CD11b/CD18 that can be mobilized to leukocyte surface and hinder the complete blockade of CD11b/CD18 with specific antibodies4, or because it is crucial to have over 90% occupancy of active integrin receptors by inhibitors for suppression of leukocyte recruitment5. Moreover, unexpected adverse effects of antibodies against β2 integrins also prohibited the use of such blocking agent in clinical trials6. Recently, Maiguel and colleagues2 reported a new approach that involves the pharmacological activation, rather than blockade of CD11b/CD18 by small molecules, which has enlightened as promising treatment strategy for inflammatory diseases. Figure 1 Schematic showing integrin antagonists and agonists differ in their ability to reduce inflammatory disease. Integrin antagonists (central panel), such as blocking mAbs, prevent leukocyte adhesion to the inflamed endothelium, thereby reducing leukocyte ... Maiguel and colleagues2 successfully identified several potent small-molecule agonists (termed as leukadherins), which specifically target and enhance CD11b/CD18-dependent cell adhesion to fibrinogen or inflamed endothelium, leading to reduction of leukocyte migration and recruitment in both in vitro and in vivo models (Figure 1). Their data provide scientific insight that instead of identification of blocking agents for integrin, discovery of potent integrin CD11b/CD18 agonists could also be another effective strategy in reducing leukocyte infiltration and subsequent inflammation in humans. Through the high-throughput screening (HTS) of a chemical library (>100 000 small molecules), three leukadherin compounds were identified to target the ligand-binding αA domain and allosterically stabilize the active conformation of CD11b/CD18. In addition, leukadherins enhanced CD11b/CD18-dependent cell adhesion and reduced leukocyte motility, which led to a substantial reduction in leukocyte transendothelial migration and recruitment into inflamed tissues. Several clinically relevant diseases models such as thioglycolate-induced peritonitis mice model, arterial balloon injury rat model and zebra-fish tailfin injury model were adopted to demonstrate the physiologically relevant settings for anti-inflammatory effects of leukadherins, The results suggest that leukadherins are therapeutic lead compounds for future optimization. Most importantly, a comparison between M1/70 mAb, a well-characterized CD11b/CD18 antibody7, and one of these compounds revealed that leukadherin shows better efficacy and well preserves organ function upon inflammatory injury. Collectively, Maiguel's data suggested that integrin-specific, small-molecule agonists represent an effective pharmacological approach for the treatment of inflammatory and autoimmune diseases. The phenotype of reduced migration and recruitment of inflammatory cells and cellular adhesion due to the increase of constitutively active integrins was firstly demonstrated in knock-in mice expressing constitutively active mutants of the integrins8. However, it is not known whether the activation of wild-type integrin receptors in normal animals could have a similar phenotype in vivo and could reduce inflammation in physiologically relevant disease models. In this respect, Maiguel and colleagues highlighted the use of chemical-biological approach in demonstrating the endogenous, wild-type integrin protein perturbed by a specific small-molecule agonist, and provided an appropriate channel to analyze the effects of integrin activation on cellular functions in vivo. On the other hand, treatment of integrin agonists in vivo would increase adhesion of inflammatory cells to the vascular endothelium that may lead to vascular damage and leakage. Although Maiguel and colleagues found no systemic signs of vascular injury or leakage from the animals administrated with compounds for more than 3 months, the bio-markers for atherosclerosis can also be monitored in animals treated with integrin agonists for a longer treatment duration9. Now, the identification of potent anti-inflammatory compounds has become a hot topic in Chinese herbal drug research10, therefore, Chinese medicinal herbs may serve as a potential source for the identification of novel integrin agonists in the future.

Novel Oligomeric Proanthocyanidin Derivatives Interact with Membrane Androgen Sites and Induce Regression of Hormone-Independent Prostate Cancer

Prostate cancer is the most common malignancy among men in Western societies, and current therapeutic approaches are evolving to manage growth, recurrence, and mortality neoplasia. Membrane androgen receptors (mARs) have been characterized in human prostate cancer, being preferentially expressed in tumor rather than benign gland areas. Furthermore, mAR agonists (protein-conjugated testosterone) decrease in vitro prostate cancer cell growth and induce apoptosis, whereas in vivo they regress growth of tumor xenografts alone or in combination with taxane drugs. In this respect, targeting mARs might be a novel therapeutic approach in prostate cancer. In our search for new small-molecule ligands of mAR, we report that flavanol dimers B1-B4 (oligomeric procyanidins) decrease in vitro growth of the androgen-sensitive (LnCaP) and androgen-resistant (DU145) human prostate cancer cell lines in the following order: B3 = B4 > B2 ≫ B1 (LnCaP) and B2 ≫ B3 = B4 ≫ B1 (DU145). Some of these analogs were previously shown to trigger signaling cascades similar to testosterone-bovine serum albumin (BSA) conjugate. Galloylation does not confer an additional advantage; however, oleylation increases the dimers' antiproliferative potency by a factor of 100. In addition, we report that B2, oleylated or not, displaces testosterone from mARs with an IC(50) value at the nanomolar range and induces DU145 tumor xenograft regression by 50% (testosterone-BSA 40%). In this respect, oleylated B2 is a potent small-molecule agonist of mAR and could be a novel therapeutic agent for advanced prostate cancer, especially when taking into account the absence of androgenic actions and (liver) toxicity.

Novel Alpha-7 Nicotinic Acetylcholine Receptor Agonists Containing a Urea Moiety: Identification and Characterization of the Potent, Selective, and Orally Efficacious Agonist 1-[6-(4-Fluorophenyl)pyridin-3-yl]-3-(4-piperidin-1-ylbutyl) Urea (SEN34625/WYE-103914)

Alpha-7 nicotinic acetylcholine receptor (alpha7 nAChR) agonists are promising therapeutic candidates for the treatment of cognitive impairment. We report a series of novel, potent small molecule agonists (4-18) of the alpha7 nAChR deriving from our continuing efforts in the areas of Alzheimer's disease and schizophrenia. One of the compounds of the series containing a urea moiety (16) was further shown to be a selective agonist of the alpha7 nAChR with excellent in vitro and in vivo profiles, brain penetration, and oral bioavailability and demonstrated in vivo efficacy in multiple behavioral cognition models. Structural modifications leading to the improved selectivity profile and the biological evaluation of this series of compounds are discussed.

Discovery of 3-Aryl-4-isoxazolecarboxamides as TGR5 Receptor Agonists

A series of 3-aryl-4-isoxazolecarboxamides identified from a high-throughput screening campaign as novel, potent small molecule agonists of the human TGR5 G-protein coupled receptor is described. Subsequent optimization resulted in the rapid identification of potent exemplars 6 and 7 which demonstrated improved GLP-1 secretion in vivo via an intracolonic dose coadministered with glucose challenge in a canine model. These novel TGR5 receptor agonists are potentially useful therapeutics for metabolic disorders such as type II diabetes and its associated complications.

SAR and biological evaluation of SEN12333/WAY-317538: Novel alpha 7 nicotinic acetylcholine receptor agonist

Alpha 7 nicotinic acetylcholine receptor (α 7 nAChR) agonists are promising therapeutic candidates for the treatment of cognitive impairment associated with a variety of disorders including Alzheimer’s disease and schizophrenia. Alpha 7 nAChRs are expressed in brain regions associated with cognitive function, regulate cholinergic neurotransmission and have been shown to be down regulated in both schizophrenia and Alzheimer’s disease. Herein we report a novel, potent small molecule agonist of the alpha 7 nAChR, SEN12333/WAY-317538. This compound is a selective agonist of the α 7 nAChR with excellent in vitro and in vivo profiles, excellent brain penetration and oral bioavailability, and demonstrates in vivo efficacy in multiple behavioural cognition models. The SAR and biological evaluation of this series of compounds are discussed.

Toward small-molecule agonists of TNF receptors

Development of small-molecule agonists against members of the tumor necrosis factor (TNF) receptor superfamily remains a considerable challenge. Presentation of ligand-derived peptides on a trimeric scaffold may point the way toward development of potent small-molecule agonists against this biologically important protein superfamily.

Synthesis of a high-affinity Fluorescent PPARγ ligand for high-throughput fluorescence polarization assays

Members of the peroxisome proliferator activated receptor (PPAR) family of transcription factors are under investigation as molecular targets for the treatment of numerous diseases including Alzheimer's, asthma, atherosclerosis, inflammation, multiple sclerosis, cancer, and diabetes. We employed the X-ray crystal structure of the PPARγ subtype complexed with the potent small molecule agonist GI262570 (farglitazar) to design and synthesize a novel fluorescent and high-affinity probe for homogeneous and high-throughput fluorescent polarization (FP) assays. Examination of this X-ray structure revealed that the phenyl carbon atom meta to the oxazole moiety of GI262570 is exposed to solvent at the bottom of a narrow protein cavity. A derivative of GI262570 was synthesized bearing a linear phenylacetylene-derived side chain comprising propargylamine coupled to fluorescein. This fluorescent analogue was designed to project the fluorophore into the adjacent protein cavity with minimal effects on receptor affinity and maximal effects on fluorescence polarization properties. The recombinant PPARγ ligand binding domain protein bound tightly and specifically to this probe with K d=61±14 nM as determined by FP measurements. Competition binding assays with known PPARγ ligands provided K i values that were highly correlated with analogous values obtained by scintillation proximity (SP) assays. This fluorescent PPARγ probe enables high-throughput and homogenous FP assays for the identification of novel endogenous and exogenous PPARγ ligands, and this rational ligand design approach may be applied to other therapeutically important members of the nuclear hormone receptor superfamily.