Cell-based Reporter Gene Assay Research Articles

A mechanism of action-reflective, dual cell-based bioassay for determining the bioactivity of sclerostin-neutralizing antibodies

Osteoporosis is a major threat to the elderly worldwide. The Wnt signaling pathway plays a critical role in bone development and homeostasis. Sclerostin, a Wnt ligand inhibitor, competes with Wnt ligands for low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) on osteoblasts, thereby suppressing bone formation. Sclerostin-neutralizing monoclonal antibodies (mAbs) have emerged as a potential bone-forming therapy for osteoporosis. A cell-based bioassay which determines the relative activity of a product, related to its mechanism of action, is of great importance from drug discovery to quality control and batch release. Currently used cell-based bioassays for sclerostin-neutralizing mAbs usually use Wnt1 or Wnt3a to stimulate the Wnt pathway; sclerostin is a direct inhibitor of Wnt1 but not Wnt3a. Wnt1 is a highly hydrophobic protein that binds to the producing cell membrane and acts in a juxtacrine manner to stimulate the Wnt pathway in neighboring cells. Bioassays for drugs that induce Wnt1 signaling should be performed in a juxtacrine manner. Here, we present a mechanism of action-reflective, dual cell-based reporter gene assay. In this assay, Wnt1 producer cells are co-cultured with cells containing the Wnt reporter genes, Wnt1 on the producer cells activates the Wnt signaling pathway in the reporter cells that are in direct cell-to-cell contact, and sclerostin-neutralizing mAbs specifically and effectively antagonize the sclerostin-mediated Wnt reporter gene suppression. This bioassay demonstrates good specificity, accuracy, linearity, and precision and is suitable for quality control, stability testing, batch release, and biosimilarity assessment of sclerostin-neutralizing mAbs.

Development of a reliable cell-based reporter gene assay to measure the bioactivity of anti-HER2 therapeutic antibodies

Human epidermal growth factor receptor 2 (HER2) is a key player in the pathogenesis and progression of breast cancer and is currently a primary target for breast cancer immunotherapy. Bioactivity determination is necessary to guarantee the safety and efficacy of therapeutic antibodies targeting HER2. Nevertheless, currently available bioassays for measuring the bioactivity of anti-HER2 mAbs are either not representative or have high variability. Here, we established a reliable reporter gene assay (RGA) based on T47D-SRE-Luc cell line that expresses endogenous HER2 and luciferase controlled by serum response element (SRE) to measure the bioactivity of anti-HER2 antibodies. Neuregulin-1 (NRG-1) can lead to the heterodimerization of HER2 on the cell membrane and induce the expression of downstream SRE-controlled luciferase, while pertuzumab can dose-dependently reverse the reaction, resulting in a good dose-response curve reflecting the activity of the antibody. After optimizing the relevant assay parameters, the established RGA was fully validated based on ICH-Q2 (R1), which demonstrated that the method had excellent specificity, accuracy, precision, linearity, and stability. In summary, this robust and innovative bioactivity determination assay can be applied in the development and screening, release control, biosimilar assessment and stability studies of anti-HER2 mAbs.

Discovery of Disubstituted Carboranes as Inhibitors of Heat Shock Protein 90-Heat Shock Factor 1 Interaction.

Efficient synthesis of disubstituted para- and ortho-carboranes (2 and 3, respectively) was achieved. Among the compounds synthesized, 3e showed potent suppression of hypoxia-inducible factor 1 (HIF-1) transcriptional activity under hypoxia by a cell-based reporter gene assay. Detailed mechanism-of-action studies revealed that 3e reduced the stability of heat shock protein (HSP) 90 client proteins such as CDK4, AKT, and cyclin D1 by inhibiting HSP90 chaperone activity but did not induce a heat shock response (HSR), which may cause drug resistance. Furthermore, 3e inhibited the interaction between HSP90 and heat shock factor 1 (HSF1), resulting in reducing HSF1 protein stability and thereby suppressing the transcription of heat shock proteins.

Structural insights into the HDAC4-MEF2A-DNA complex and its implication in long-range transcriptional regulation.

Class IIa Histone deacetylases (HDACs), including HDAC4, 5, 7and 9, play key roles in multiple important developmental and differentiation processes. Recent studies have shown that class IIa HDACs exert their transcriptional repressive function by interacting with tissue-specific transcription factors, such as members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. However, the molecular mechanism is not well understood. In this study, we determined the crystal structure of an HDAC4-MEF2A-DNA complex. This complex adopts a dumbbell-shaped overall architecture, with a 2:4:2 stoichiometry of HDAC4, MEF2A and DNA molecules. In the complex, two HDAC4 molecules form a dimer through the interaction of their glutamine-rich domain (GRD) to form the stem of the 'dumbbell'; while two MEF2A dimers and their cognate DNA molecules are bridged by the HDAC4 dimer. Our structural observations were then validated using biochemical and mutagenesis assays. Further cell-based luciferase reporter gene assays revealed that the dimerization of HDAC4 is crucial in its ability to repress the transcriptional activities of MEF2 proteins. Taken together, our findings not only provide the structural basis for the assembly of the HDAC4-MEF2A-DNA complex but also shed light on the molecular mechanism of HDAC4-mediated long-range gene regulation.

Synthesis and Evaluation of Small Molecule Inhibitors of the Androgen Receptor N-Terminal Domain.

The androgen receptor (AR) is central to prostate cancer pathogenesis and has been extensively validated as a drug target. However, small-molecule anti-androgen therapies remain limited due to resistance and will eventually fail to suppress tumor growth, resulting in progression to castration-resistant prostate cancer (CRPC). The intrinsically disordered N-terminal domain (NTD) is crucial for AR transactivation and has been investigated as a suitable target in the presence of ligand binding domain mutations. A screening campaign identified biaryl isoxazole compound 7 as a weak inhibitor of the AR NTD. A library of biaryl analogues were synthesized, and their biological activities were assessed in a VCaP cell-based luciferase reporter gene assay. A structure-activity relationship (SAR) study revealed that indazole analogue 16 exhibited increased potency and favorable physicochemical properties with a benchmarked pharmacokinetic profile, providing a suitable starting point for further optimization of 16 as a CRPC therapeutic in the presence of AR mutations.

St. John's wort formulations induce rat CYP3A23-3A1 independent of their hyperforin content.

The pregnane X receptor (PXR) is a ligand activated regulator of cytochrome P450 (CYP)3A enzymes. Among the ligands of human PXR is hyperforin, a constituent of St John's wort (SJW) extracts and potent inducer of human CYP3A4. It was the aim of this study to compare the effect of hyperforin and SJW formulations controlled for its content on CYP3A23-3A1 in rats. Hyperiplant® was used as it contains a high hyperforin content and Rebalance® because it is controlled for a low hyperforin content. In silico analysis revealed a weak hyperforin-rPXR binding affinity, which was further supported in cell-based reporter gene assays showing no hyperforin-mediated reporter activation in presence of rPXR. However, cellular exposure to Hyperiplant® and Rebalance® transactivated the CYP3A-reporter 3.8 fold and 2.8 fold, respectively, and they induced Cyp3a23-3a1 mRNA expression in rat hepatoma cells compared to control 48-fold and 18-fold, respectively. In Wistar rats treated for 10 days with 400 mg/kg of Hyperiplant® we observed 1.8-times the Cyp3a23-3a1 mRNA expression, a 2.6-fold higher CYP3A23-3A1 protein amount and a 1.6-fold increase in activity compared to control. For Rebalance® we only observed a 1.8-fold hepatic increase of CYP3A23-3A1 protein compared to control animals. Even though there are differing effects on rCyp3a23-3a1/CYP3A23-3A1 in rat liver reflecting the hyperforin content of the SJW extracts, the modulation is most likely not linked to an interaction of hyperforin with rPXR. Significance Statement Treatment with St John's wort (SJW) has been reported to affect CYP3A expression and activity in rats. Our comparative study further supports this finding but shows that the PXR-ligand hyperforin is not the driving force for changes in rat CYP3A23-3A1 expression and function in vivo and in vitro Importantly, CYP3A induction mimics findings in humans but our results suggest that another so far unknown constituent of SJW is responsible for the expression and function modifying effects in rat liver.

Comparison of Two Clinical Laboratory Assays for Measuring Serum Adalimumab and Antibodies to Adalimumab.

Adalimumab is a fully human monoclonal antibody developed against tumor necrosis factor (TNF), used for the treatment of autoimmune and chronic inflammatory diseases. Immunogenicity to this drug may lead to therapeutic failure. Various laboratory assays are used for measuring serum adalimumab and anti-drug antibodies (ADA) to adalimumab, for therapeutic monitoring and evaluation of clinical non-responsiveness. This study compared the performance of 2 clinical assays used by different reference laboratories. In total, 120 residual clinical samples were tested at both laboratories. A sandwich ELISA for adalimumab detecting free drug and a bridging ELISA capable of detecting both free and bound ADA were performed at the Mayo Clinic. A functional cell-based reporter gene assay (RGA) was used at ARUP Laboratories for measuring bioactive serum drug concentrations, and neutralizing ADA. Seventy-eight samples had measurable concentrations of adalimumab by both methods and yielded a correlation coefficient r = 0.93, slope = 0.886, and intercept = 0.950. Overall agreement of 92.5% was observed between the assays, with most discrepant drug results being attributed to a higher positivity rate with ELISA (8/9). One outlier positive with RGA and negative with ELISA was confirmed by LC-MS/MS to be attributed to infliximab. Overall agreement of 79.2% was observed between the ADA assays. Differences in ADA results may be due to the bridging ELISA detecting total ADA (free, drug-bound, neutralizing, and non-neutralizing), while RGA detects free, neutralizing ADA only. Although the assays are fundamentally different, the results show significant concordance between the clinically validated tests performed in different laboratories.

Discovery of novel triazine derivatives as potent retinoic acid receptor-related orphan receptor γt (RORγt) inverse agonists

Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) has been exploited as a promising target for the new small molecule therapeutics to treat inflammatory and autoimmune diseases via modulating the interleukin-17 (IL-17) production by T helper 17 (Th17) cells. Herein, we reported a series of triazine-based derivatives as novel RORγt inverse agonists. By screening of our in-house compound library, the hit compound 1 was identified with weak RORγt inhibitory activity. Subsequently, we engineered detailed structural modifications to explore the structure-activity relationships (SARs) of triazines derivatives, which led to discovery of a number of potent RORγt inverse agonists with IC50 values in the range of 7 nM–50 nM in RORγt dual FRET assay. Among them, compound 14g displayed potent RORγt inverse agonistic activity with an IC50 value of 22.9 nM in dual FRET assay. In a cell-based reporter gene assay, compound 14g showed an IC50 value of 0.428 μM and maximum inhibition rate of 108.9%. Compound 14g also exhibited good metabolic stability and a decent pharmacokinetic profile with a low clearance (CL = 0.229 L/h/kg) and a reasonable oral exposure (AUC0-Last = 5058 ng/mL*h). Most importantly, 14g alleviated the severity of imiquimod-induced psoriasis in mice. Taken together, triazine-based derivatives represent a new chemical class of RORγt inverse agonists as potential therapeutic agents against autoimmune diseases.

Abstract 5657: HK010, a novel anti-PDL1 × CD137 bispecific antibody, exhibits potent anti-tumor immunity and low toxicity

Abstract Background: Checkpoint inhibitors targeting the PD-1/PD-L1 have changed the treatment and prognosis for patients with advanced solid tumors. There remains a unmet need for additional therapies due to treatment resistance. Tumor-necrosis superfamily members, such as CD137 and CD40, have been shown to synergize with the checkpoint inhibitors in preclinical studies. However, agonists antibodies have exhibited limited clinical activity for severe liver toxicity. Here we have developed a Fc-silenced bispecific antibody targeting PD-L1 and CD137, HK010, which could overcome these limitations by tumor-directed T-cell activation and checkpoint blockade. Methods: The antigen binding specificity and affinity of HK010 were determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) and flow cytometry. The functional activity of HK010 was determined using in vitro cell-based assays and reporter gene assay. Transgenic mice expressing human PD-L1 and 4-1BB were transplanted with human PD-L1-expressing murine MC38 cells to assess in vivo antitumor activity. Cytokine release assay was conducted by incubating HK010 with peripheral blood mononuclear cells (PBMCs) from healthy donors (n=6). The pharmacokinetic (PK) behavior and safety profiles of HK010 were characterized in cynomolgus monkeys. Results: HK010 is a IgG4 PD-L1 × 4-1BB bsAb, which was designed to maintain a high affinity for human PD-L1 and a low affinity for human 4-1BB (KD PD-L1: 2.27 nM; KD 4-1BB: 493 nM) to achieve strong blocking of PD-1/PD-L1 and appropriate agonism of 4-1BB. HK010 retained full blockade activity on PD-1/PD-L1 signaling and enhances T-cell proliferation and IFN-γ production in vitro primary cell assays and Mixed lymphoid reaction (MLR). In addition, it led to a dose-dependent increase in the CD137-driven NFκB reporter gene activation through the bridging of PD-L1 on target cells and CD137 on effector cells. In humanized PD-L1/4-1BB transgenic mice bearing with MC38/hPDL1 tumor, HK010 showed robust single agent anti-tumor activity and induced durable antigen-specific immunological memory that prevents the growth of the same tumor cells in the mice re-challenged. There were no signs of toxicity in human cytokine release assay for HK010. Toxicology data in cynomolgus monkeys showed that HK010 was well-tolerated up to 150 mg/kg (MTD≥150mg/kg) dose without systemic toxicity. Furthermore, HK010 was confirmed to be highly safe in the 5-week repeated-dose toxicity study with no observed treatment-related mortality, abnormality in hematological indexes, important organs. Conclusion: These data demonstrate that HK010, an anti-PD-L1 × CD137 bispecific antibody, may exert a strong localized anti-tumor therapeutic efficacy with a low risk of liver toxicity through tumor-directed T-cell activation and checkpoint blockade in tumors. Citation Format: Liangwei Li, Wenting Liu, Guodong Shen, Dayan Zhang, Weiming Zhou, Fengrong Wang, Xiaoli Zeng, Yang Huang, Liansheng Cheng. HK010, a novel anti-PDL1 × CD137 bispecific antibody, exhibits potent anti-tumor immunity and low toxicity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5657.

Development of a reporter gene assay for antibody dependent cellular cytotoxicity activity determination of anti-rabies virus glycoprotein antibodies.

Rabies is a viral disease that is nearly 100% fatal once clinical signs and symptoms develop. Post-exposure prophylaxis can efficiently prevent rabies, and antibody (Ab) induction by vaccination or passive immunization of human rabies immunoglobulin (HRIG) or monoclonal antibodies (mAbs) play an integral role in prevention against rabies. In addition to their capacity to neutralize viruses, antibodies exert their antiviral effects by antibody-dependent cellular cytotoxicity (ADCC), which plays an important role in antiviral immunity and clearance of viral infections. For antibodies against rabies virus (RABV), evaluation of ADCC activity was neglected. Here, we developed a robust cell-based reporter gene assay (RGA) for the determination of the ADCC activity of anti-RABV antibodies using CVS-N2c-293 cells, which stably express the glycoprotein (G) of RABV strain CVS-N2c as target cells, and Jurkat cells, which stably express FcγRⅢa and nuclear factor of activated T cells (NFAT) reporter gene as effector cells (Jurkat/NFAT-luc/FcγRⅢa cells). The experimental parameters were carefully optimized, and the established ADCC assay was systematically validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2 guideline. We also evaluated the ADCC activity of anti-RABV antibodies, including mAbs, HRIG, and vaccine induced antisera, and found that all test antibodies exhibited ADCC activity with varied strengths. The established RGA provides a novel method for evaluating the ADCC of anti-RABV antibodies.

A conserved domain of Drosophila RNA-binding protein Pumilio interacts with multiple CCR4–NOT deadenylase complex subunits to repress target mRNAs

Pumilio is a sequence-specific RNA-binding protein that controls development, stem cell fate, and neurological functions in Drosophila. Pumilio represses protein expression by destabilizing target mRNAs in a manner dependent on the CCR4–NOT deadenylase complex. Three unique repression domains in the N-terminal region of Pumilio were previously shown to recruit CCR4–NOT, but how they do so was not well understood. In this study, we identified the motifs that are necessary and sufficient for the activity of the third repression domain of Pumilio, designated RD3, which is present in all isoforms and has conserved regulatory function. We identified multiple conserved regions of RD3 that are important for repression activity in cell-based reporter gene assays. Using yeast two-hybrid assays, we show that RD3 contacts specific regions of the Not1, Not2, and Not3 subunits of the CCR4–NOT complex. Our results indicate that RD3 makes multivalent interactions with CCR4–NOT mediated by conserved short linear interaction motifs. Specifically, two phenylalanine residues in RD3 make crucial contacts with Not1 that are essential for its repression activity. Using reporter gene assays, we also identify three new target mRNAs that are repressed by Pumilio and show that RD3 contributes to their regulation. Together, these results provide important insights into the mechanism by which Pumilio recruits CCR4–NOT to regulate the expression of target mRNAs.

Characterization of functionally deficient SIM2 variants found in patients with neurological phenotypes.

Single-minded 2 (SIM2) is a neuron-enriched basic Helix–Loop–Helix/PER–ARNT–SIM (bHLH/PAS) transcription factor essential for mammalian survival. SIM2 is located within the Down syndrome critical region (DSCR) of chromosome 21, and manipulation in mouse models suggests Sim2 may play a role in brain development and function. During the screening of a clinical exome sequencing database, nine SIM2 non-synonymous mutations were found which were subsequently investigated for impaired function using cell-based reporter gene assays. Many of these human variants attenuated abilities to activate transcription and were further characterized to determine the mechanisms underpinning their deficiencies. These included impaired partner protein dimerization, reduced DNA binding, and reduced expression and nuclear localization. This study highlighted several SIM2 variants found in patients with disabilities and validated a candidate set as potentially contributing to pathology.

Artificial infiltration in drinking water production: Addressing chemical hazards using effect-based methods

Artificial infiltration is an established managed aquifer recharge method that is commonly incorporated into drinking water processes. However, groundwater sourced from this type of purification method is prone to contamination with chemical hazards. Such an instance was previously shown at a Swedish DWTP where the river water was contaminated by hazardous chemicals during artificial infiltration. Further, there remains a paucity of research studying the quality of drinking water following this type of treatment from an effect-based bioanalytical perspective. In the current study, an effect-based assessment for chemical hazards was conducted for a Swedish drinking water system comprised of two DWTPs fed artificially-infiltrated river water. In this system, artificial infiltration of the river water takes approximately six to eight months. A sampling event was conducted in the autumn season and the samples were enriched by solid phase extraction. A panel of cell-based reporter gene assays representing several toxicity pathways was selected: oxidative stress response (Nrf2 activity), aryl hydrocarbon receptor (AhR) activation, and hormone receptor-mediated effects (estrogen receptor [ER], androgen receptor [AR]). AhR and ER bioactivities were detected in samples collected from the river intake and in the open-air infiltration basins prior to artificial infiltration. However, the AhR activity decreased and ER activity was effectively removed following artificial infiltration. In the Nrf2 and AR assays, no bioactivities above cut-off levels were detected in any samples collected along the entire treatment process of the drinking water production from source to tap. Using a suite of bioassays, the current study highlighted the effectiveness of artificial infiltration in reducing bioactive compounds in this raw river water. Although artificial infiltration is a common purification method in drinking water production, the limited number of effect-based studies evaluating the effectiveness of this method emphasizes the need for further research to better understand the risks and benefits of this water treatment process.

ROR\u03b3t agonist enhances anti-PD-1 therapy by promoting monocyte-derived dendritic cells through CXCL10 in cancers

BackgroundThe overall response rate to checkpoint blockade remains unsatisfactory, partially due to the immune-suppressive tumor microenvironment. A retinoic acid-related orphan receptor γt (RORγt) agonist (LYC-55716) is currently used in clinical trials combined with anti-PD-1, but how the Th17 cell transcription factor RORγt enhances antitumor immunity of PD-1 in the tumor microenvironment remains elusive.MethodsThe expression of mRNA was analyzed using qPCR assays. Flow cytometry was used to sort and profile cells. Cell migration was analyzed using Transwell assays. Biacore was used to determine the binding affinity to the RORγt protein. The RORγt GAL4 cell-based reporter gene assay was used to measure activity in the RORγt driven luciferase reporter gene expression.ResultsWe designed a potent and selective small-molecule RORγt agonist (8-074) that shows robust antitumor efficacy in syngeneic tumor models and improves the efficacy of anti‑PD‑1 in a murine lung cancer model. RORγt agonist treatment increased intratumoral CD8+ T cells, which were correlated with CXCL10 and monocyte-derived dendritic cells (MoDCs). In addition, the RORγt agonist promoted Type 17 T cell migration by upregulating CCL20 and CCR6 expression, and Type 17 T cell tumor infiltration. CCL20 induces MoDCs migration, and CXCL10 derived from MoDCs promotes CD8+ T cell migration.ConclusionOur results revealed that the RORγt agonist improved the efficacy of anti-PD-1. The RORγt agonist increased the migration of MoDCs, which increased the local levels of CXCL10, thus promoting CD8+ T cell tumor infiltration. Our findings provide the mechanistic insights implicating the RORγt agonist in immunotherapy and offer a strategy for targeting the RORγt agonist to improve PD-1 antibody efficacy in cancers.

The Influence of Intracellular Glutathione Levels on the Induction of Nrf2-Mediated Gene Expression by α-Dicarbonyl Precursors of Advanced Glycation End Products.

α-Dicarbonyl compounds, particularly methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG), are highly reactive precursors for the formation of advanced glycation end products (AGEs). They are formed in vivo and during food processing. This study aimed to investigate the role of intracellular glutathione (GSH) levels in the induction of Nrf2-mediated gene expression by α-dicarbonyl compounds. The reactions between α-dicarbonyl compounds (MGO, GO, and 3-DG) and GSH were studied by LC-MS in a cell-free system. It was shown that these three α-dicarbonyl compounds react instantaneously with GSH, with the GSH-mediated scavenging decreasing in the order MGO > GO > 3DG. Furthermore, in a cell-based reporter gene assay MGO, GO, and 3-DG were able to induce Nrf2-mediated gene expression in a dose-dependent manner. Modulation of intracellular GSH levels showed that the cytotoxicity and induction of the Nrf2-mediated pathway by MGO, GO and 3-DG was significantly enhanced by depletion of GSH, while a decrease in Nrf2-activation by MGO and GO but not 3-DG was observed upon an increase of the cellular GSH levels. Our results reveal subtle differences in the role of GSH in protection against the three typical α-dicarbonyl compounds and in their induction of Nrf2-mediated gene expression, and point at a dual biological effect of the α-dicarbonyl compounds, being reactive toxic electrophiles and -as a consequence- able to induce Nrf2-mediated protective gene expression, with MGO being most reactive.

Front Cover: Andrographolide Derivatives Target the KEAP1/NRF2 Axis and Possess Potent Anti‐SARS‐CoV‐2 Activity (ChemMedChem 5/2022)

The Front Cover illustrates the natural product andrographolide, which modulates the abundance of the transcription factor NRF2, a substrate of the E3 ligase KEAP1. Previous studies identified that this drug possessed anti-SARS-CoV-2 activity, but the mechanism of action remained unclear. The authors designed and synthesized novel andrographolide derivatives with a functional site to fine-tune physicochemical properties and for linker attachment. The team assayed this new set of compounds in a cell-based NRF2 reporter gene assay and determined their ability to decrease infectivity of virus-treated Vero-E6 cells. Data showed that NRF2 activation by compounds and inhibition of SARS-CoV-2 replication correlated well. The study opens new avenues to investigate natural products that target the KEAP1/NRF2 axis as anti-SARS-CoV-2 agents. More information can be found in the Research Article by Christian Steinebach et al.

SUVN-D4010: A serotonin-4 receptor partial agonist for the treatment of cognitive disorders.

Memory impairments are clinical symptoms of a wide range of neurodegenerative diseases, and natural physiological process such as ageing. SUVN-D4010 is an investigational clinical candidate which acts by modulating 5-HT4 receptors. Experimental evidences from animals and humans suggest that 5-HT4 receptor activation holds promise for the treatment of cognitive impairment. Additionally, activation of 5-HT4 receptor also modulates mood. The present investigation is to characterize SUVN-D4010 and evaluate its drug-like properties. SUVN-D4010 has been studied for its affinity and functional activity at 5-HT4 receptor using in vitro methods. ADME properties were evaluated using in vitro and in vivo methods. SUVN-D4010 was studied for its effects on learning and memory using social and object recognition tasks. The effects on the neurotransmitters were monitored using the brain microdialysis technique in rats. The effects on the beta amyloid processing were studied using in vitro and in vivo systems. The safety and tolerability was evaluated in both animals and humans. SUVN-D4010 showed potent affinity towards 5-HT4 receptor when tested using radioligand binding and cell based reporter gene assay. SUVN-D4010 has minimal binding over 70 target sites (at 1 µM, Caliper) including GPCRs, transporters, ion channels, peptides, steroids, second messengers, growth factors and prostaglandins. SUVN-D4010 is orally bioavailable with good brain penetration and CSF concentrations. The likelihood of drug-drug interaction of SUVN-D4010 as an inhibitor/ inducer is remote. No unique metabolites were identified in non-clinical and clinical samples. SUVN-D4010 showed procognitve effects at doses ranging from 0.3 to 3 mg/kg, p.o. SUVN-D4010 significantly increased extracellular acetylcholine levels as a standalone, or treatment adjunct to donepezil. SUVN-D4010 promoted non-amyloidogenic APPα processing and reduced toxic Aβ load. SUVN-D4010 was found to be safe and well tolerated in healthy human volunteers. SUVN-D4010 is a potent, selective, orally bioavailable and brain penetrant 5-HT4 receptor partial agonist with potential utility in treatment of cognitive disorders.

Functionally diverse heteromeric traps for ligands of the transforming growth factor-\u03b2 superfamily

Ligands of the transforming growth factor-β (TGF-β) superfamily are important targets for therapeutic intervention but present challenges because they signal combinatorially and exhibit overlapping activities in vivo. To obtain agents capable of sequestering multiple TGF-β superfamily ligands with novel selectivity, we generated soluble, heterodimeric ligand traps by pairing the extracellular domain (ECD) of the native activin receptor type IIB (ActRIIB) alternately with the ECDs of native type I receptors activin receptor-like kinase 4 (ALK4), ALK7, or ALK3. Systematic analysis of these heterodimeric constructs by surface plasmon resonance, and comparison with their homodimeric counterparts, revealed that each type I receptor partner confers a distinct ligand-binding profile to the heterodimeric construct. Additional characterization in cell-based reporter gene assays confirmed that the heterodimeric constructs possessed different profiles of signaling inhibition in vitro, which translated into altered patterns of pharmacological activity when constructs were administered systemically to wild-type mice. Our results detail a versatile platform for the modular recombination of naturally occurring receptor domains, giving rise to inhibitory ligand traps that could aid in defining the physiological roles of TGF-β ligand sets or be directed therapeutically to human diseases arising from dysregulated TGF-β superfamily signaling.

A systematic approach to metabolic characterization of thyroid-disrupting chemicals and their in vitro biotransformants based on prediction-assisted metabolomic analysis

Thyroid-disrupting compounds (TDCs) are chemicals that modify thyroid gland function and disrupt hormonal homeostasis. Like other endocrine-disrupting chemicals (EDCs), TDCs often show altered activities following post-metabolic modification via endogenous enzymatic reaction.Hence, we developed evaluation system consisting of (1) in vitro metabolic reaction module, (2) high-resolution mass-spectrometry, and (3) human cell-based reporter gene assay. We developed the reaction module using rat S9 fraction where levothyroxine (T4) as a model compound, was subjected to phase-I or phase-I+II biotransformation. The metabolic profiles of the biotransformants were systematically configured based on in-silico prediction of potential products and experimental validation using liquid-chromatography Orbitrap mass-spectrometry. Thyroid agonistic activities of the biotransformants were evaluated by thyroid receptor-mediated stably transfected transcriptional activation assay using hTRE_HeLa cells.Indeed, we detected the increased activities following metabolic conversion of T4 in a dose-dependent manner. Note that the activity by phase-I+II reaction was much greater than by phase-I reaction (3.8-fold increase). Subsequently, we explored metabolic signatures, which potentially contributed to the hyperactivity by phase-I+II reaction. A total of 77 metabolic features were annotated based on the in-silico prediction, which included biotransformants with deiodination and conjugation. The glucuronide-conjugated form was found at the highest fold-increase (970-fold increase) whereas marginal increases were determined in the deiodinized forms (1.6-fold increase in T3 and 2.0-fold increase in rT3). Further, the systematic approach was evaluated and comparably analyzed by the metabolic profiles of bithionol, which is structurally related to T4. Our current result suggested the potential application of in vitro evaluation system to risk assessment of thyroid-disrupting activity.

A cell-based ribozyme reporter system employing a chromosomally-integrated 5\u2032 exonuclease gene

BackgroundBioinformatic genome surveys indicate that self-cleaving ribonucleic acids (ribozymes) appear to be widespread among all domains of life, although the functions of only a small number have been validated by biochemical methods. Alternatively, cell-based reporter gene assays can be used to validate ribozyme function. However, reporter activity can be confounded by phenomena unrelated to ribozyme-mediated cleavage of RNA.ResultsWe established a ribozyme reporter system in Escherichia coli in which a significant reduction of reporter activity is manifest when an active ribozyme sequence is fused to the reporter gene and the expression of a foreign Bacillus subtilis RNaseJ1 5′ exonuclease is induced from a chromosomally-integrated gene in the same cell.ConclusionsThe reporter system could be useful for validating ribozyme function in candidate sequences identified from bioinformatics.