Small Molecule Screening Research Articles

I.12 Modulating muscle stem cells to enhance regeneration to ameliorate DMD disease progression

Duchenne muscular dystrophy (DMD) is a lethal X-linked muscle-wasting disease. Dystrophin functions as an essential component of the large oligomeric dystrophin-glycoprotein complex (DGC). The DGC acts to connect the actin cytoskeleton of the myofiber to the surrounding extracellular matrix through the sarcolemma. In the absence of dystrophin, DGC assembly is impaired which weakens the muscle fibers rendering them highly susceptible to injury, causing accumulation of inflammation and fibrosis, and progressive loss of muscle mass and function. We discovered that dystrophin is highly expressed in activated muscle stem cells and associates with the serine-threonine kinase Mark2/Par1b, a regulator of cell polarity. In the absence of dystrophin, Mark2 is downregulated, resulting in the inability to localize the cell polarity regulator Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions and hence the generation of myogenic progenitors is significantly reduced. Our findings indicate that muscle wasting in DMD not only is caused by myofiber fragility, but also is exacerbated by impaired regeneration owing to intrinsic satellite cell dysfunction. Through a small-molecule screen, we made the discovery of a novel signal transduction pathway involving epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurka) as key regulators of asymmetric satellite cell divisions. Inhibiting EGFR causes a substantial shift from asymmetric to symmetric division modes, whereas EGF treatment increases asymmetric divisions. EGFR activation acts through Aurka to orient mitotic centrosomes, and inhibiting Aurka blocks EGF stimulation-induced asymmetric division. In vivo EGF treatment markedly activates asymmetric divisions of dystrophin-deficient satellite cells in mdx mice, increasing progenitor numbers, enhancing regeneration, and restoring muscle strength. Therefore, activating an EGFR-dependent polarity pathway promotes functional rescue of dystrophin-deficient satellite cells and enhances muscle force generation. In collaboration with Satellos Bioscience, we have been developing small molecule drugs that stimulate asymmetric division by modulating the EGFR/Aurka and other pathways. Our goal is to develop small molecule modulators of muscle stem cell asymmetric division as a treatment for DMD.

VP.65 Screening of small molecules for activation of GNE protein carrying non-catalytic site mutation based on molecular docking simulation

GNE myopathy is an autosomal recessive distal myopathy caused by pathogenic variants in <i>GNE</i> gene, which encodes a protein having two enzymatic activities, UDP-GlcNAc 2-epimerase and ManNAc kinase on sialic acid biosynthesis. We have identified a variant, c.620A>T (p.D207V), with which majority of the homozygotes are healthy, while compound heterozygotes develop myopathy. Notwithstanding, the identified three affected homozygotes had no additional variation in the <i>GNE</i> gene nor in genes in sialic acid biosynthetic pathway, while their sialylation was mildly, but certainly decreased. We hypothesized this discrepancy can be explainable by partial failure of GNE oligomerization. The constructed full-length GNE structure by Alphafold2 indicated that Asp207 locates midway between each domain and near subunit interface, but not in catalytic site. The mutant was predicted to cause no severe changes in a whole structure, though the change of polarity of the mutated residue and failure of oligomerization are hypothesized to affect intramolecular and inter-subunit ManNAc transfer between domains. Since this pathogenic variant is in non-catalytic site and did not cause drastic structural changes, ManNAc production has possibility to be pharmacologically recovered by small molecules. Here, we performed <i>in silico</i> prediction-based screening to identify the drugs that activate GNE. First, we targeted a deep narrow cavity found in the back of the allosteric site, to find small molecules that interfere with negative feedback inhibition. We then performed docking simulation of 8042 known compounds in Drugbank database to the cavity, and selected well-fitted 8 compounds. We measured the effect of the compounds on ManNAc production. However, no compounds could activate it nor repress inhibitory effect of CMP-NeuAc. In this presentation, we will discuss brand-new approach to find activators for endogenous mutated GNE protein using structural information.

Screening of Chemical Libraries Using Xenopus Embryos and Tadpoles for Phenotypic Drug Discovery.

Phenotypic drug discovery assesses the effect of small molecules on the phenotype of cells, tissues, or whole organisms without a priori knowledge of the target or pathway. Using vertebrate embryos instead of cell-based assays has the advantage that the screening of small molecules occurs in the context of the complex biology and physiology of the whole organism. Fish and amphibians are the only classes of vertebrates with free-living larvae amenable to high-throughput drug screening in multiwell dishes. For both animal classes, particularly zebrafish and Xenopus, husbandry requirements are straightforward, embryos can be obtained in large numbers, and they develop ex utero so their development can be monitored easily with a dissecting microscope. At 350 million years, the evolutionary distance between amphibians and humans is significantly shorter than that between fish and humans, which is estimated at 450 million years. This increases the likelihood that drugs discovered by screening in amphibian embryos will be active in humans. Here, we describe the basic protocol for the medium- to high-throughput screening of chemical libraries using embryos of the African clawed frog Xenopus laevis Bioactive compounds are identified by observing phenotypic changes in whole embryos and tadpoles. In addition to the discovery of compounds with novel bioactivities, the phenotypic screening protocol also allows for the identification of compounds with in vivo toxicity, eliminating early hits that are poor drug candidates. We also highlight important considerations for designing chemical screens, choosing chemical libraries, and performing secondary screens using whole mount in situ hybridization or immunostaining.

Transient targeting of BIM-dependent adaptive MCL1 preservation enhances tumor response to molecular therapeutics in non-small cell lung cancer.

Despite remarkable efficacy, targeted treatments often yield a subpopulation of residual tumor cells in part due to non-genetic adaptions. Previous mechanistic understanding on the emergence of these drug-tolerant persisters (DTPs) has been limited to epigenetic and transcriptional reprogramming. Here, by comprehensively interrogating therapy-induced early dynamic protein changes in diverse oncogene-addicted non-small cell lung cancer models, we identified adaptive MCL1 increase as a new and universal mechanism to confer apoptotic evasion and DTP formation. In detail, acute MAPK signaling disruption in the presence of genotype-based tyrosine kinase inhibitors (TKIs) prompted mitochondrial accumulation of pro-apoptotic BH3-only protein BIM, which sequestered MCL1 away from MULE-mediated degradation. A small-molecule combination screen uncovered that PI3K-mTOR pathway blockade prohibited MCL1 upregulation. Biochemical and immunocytochemical evidence indicated that mTOR complex 2 (mTORC2) bound and phosphorylated MCL1, facilitating its interaction with BIM. As a result, short-term polytherapy combining antineoplastic TKIs with PI3K, mTOR or MCL1 inhibitors sufficed to prevent DTP development and promote cancer eradication. Collectively, these findings support that upfront and transient targeting of BIM-dependent, mTORC2-regulated adaptive MCL1 preservation holds enormous promise to improve the therapeutic index of molecular targeted agents.

Biophysical Analysis of Small Molecule Binding to Viral RNA Structures.

RNA molecules are essential for carrying genetic information and regulating gene expression in most organisms including human pathogenic RNA and relate retro viruses. Targeting viral RNA (vRNA) structures provide broad opportunities to develop chemical tools to probe molecular virology and to discover novel targets for therapeutic intervention. An increasing number of RNA binding small molecules are being identified, stimulating increased interests in small molecule drug discovery for RNA targets. In this chapter, we describe protocols to characterize and robustly validate vRNA-small molecule (vRNA-sm) interactions starting from vRNA sample preparation, followed by small molecule screening against vRNA targets and finally to validating the vRNA-sm interactions via NMR spectroscopy and calorimetric titrations.

High-throughput small molecule screen identifies inhibitors of microsporidia invasion and proliferation in C. elegans.

Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia represent to humans and agricultural animals, few reliable treatment options exist. Here, we develop a high-throughput screening method for the identification of chemical inhibitors of microsporidia infection, using liquid cultures of Caenorhabditis elegans infected with the microsporidia species Nematocida parisii. We screen a collection of 2560 FDA-approved compounds and natural products, and identify 11 candidate microsporidia inhibitors. Five compounds prevent microsporidia infection by inhibiting spore firing, whereas one compound, dexrazoxane, slows infection progression. The compounds have in vitro activity against several other microsporidia species, including those known to infect humans. Together, our results highlight the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens, and provide a scalable high-throughput system for the identification and characterization of microsporidia inhibitors.

Cartilaginous Metabolomics Reveals the Biochemical-Niche Fate Control of Bone Marrow-Derived Stem Cells.

Joint disorders have become a global health issue with the growth of the aging population. Screening small active molecules targeting chondrogenic differentiation of bone marrow-derived stem cells (BMSCs) is of urgency. In this study, microfracture was employed to create a regenerative niche in rabbits (n = 9). Cartilage samples were collected four weeks post-surgery. Microfracture-caused morphological (n = 3) and metabolic (n = 6) changes were detected. Non-targeted metabolomic analysis revealed that there were 96 differentially expressed metabolites (DEMs) enriched in 70 pathways involved in anti-inflammation, lipid metabolism, signaling transduction, etc. Among the metabolites, docosapentaenoic acid 22n-3 (DPA) and ursodeoxycholic acid (UDCA) functionally facilitated cartilage defect healing, i.e., increasing the vitality and adaptation of the BMSCs, chondrogenic differentiation, and chondrocyte functionality. Our findings firstly reveal the differences in metabolomic activities between the normal and regenerated cartilages and provide a list of endogenous biomolecules potentially involved in the biochemical-niche fate control for chondrogenic differentiation of BMSCs. Ultimately, the biomolecules may serve as anti-aging supplements for chondrocyte renewal or as drug candidates for cartilage regenerative medicine.

Identification of small-molecule inhibitors of the DNA repair proteins RuvAB from Pseudomonas aeruginosa

The Holliday junction (HJ) branch migrator RuvAB complex plays a fundamental role during homologous recombination and DNA damage repair, and therefore, is an attractive target for the treatment of bacterial pathogens. Pseudomonas aeruginosa (P. aeruginosa, Pa) is one of the most common clinical opportunistic bacterial pathogens, which can cause a series of life-threatening acute or chronic infections. Here, we performed a high throughput small-molecule screening targeting PaRuvAB using the FRET-based HJ branch migration assay. We identified that corilagin, bardoxolone methyl (BM) and 10-(6ˊ-plastoquinonyl) decyltriphenylphosphonium (SKQ1) could efficiently inhibit the branch migration activity of PaRuvAB, with IC50 values of 0.40 ± 0.04 μM, 0.38 ± 0.05 μM and 4.64 ± 0.27 μM, respectively. Further biochemical and molecular docking analyses demonstrated that corilagin directly bound to PaRuvB at the ATPase domain, and thus prevented ATP hydrolysis. In contrast, BM and SKQ1 acted through blocking the interactions between PaRuvA and HJ DNA. Finally, these compounds were shown to increase the susceptibility of P. aeruginosa to UV-C irradiation. Our work, for the first time, reports the small-molecule inhibitors of RuvA and RuvB from any species, providing valuable chemical tools to dissect the functional role of each individual protein in vivo.

Small-molecule screening identifies Syk kinase inhibition and rutaecarpine as modulators of macrophage training and SARS-CoV-2 infection.

Biologically active small molecules can impart modulatory effects, in some cases, providing extended long-term memory. In a screen of biologically active small molecules for regulators of TNF induction, we identify several compounds with the ability to induce training effects on human macrophages. Rutaecarpine shows acute and long-term modulation, enhancing LPS-induced pro-inflammatory cytokine secretion and relieving LPS tolerance in human macrophages. Rutaecarpine inhibits β-glucan-induced H3K4Me3 marks at the promoters of several proinflammatory cytokines, highlighting the potential of this molecule to modulate chromosomal topology. Syk kinase inhibitor (SYKi IV), another screen hit, promotes an enhanced response to LPS similar to that previously reported for β-glucan-induced training. Macrophages trained with SYKi IV show a high degree of resistance to influenza A, multiple variants of SARS CoV-2 and OC43 coronavirus infection, highlighting a potential application of this molecule and other Syk kinase inhibitors as prophylactic treatments for viral susceptibility.

Drug Discovery Strategies for Inherited Retinal Degenerations.

Simple SummaryInherited retinal degeneration is a group of heterogeneous genetic disorders impairing vision. In these diseases, photoreceptor or retinal ganglion cells become dysfunctional and degenerate. Currently, no cures exist for these devastating blinding conditions. In this review, we will discuss targets and drug discovery strategies for inherited retinal degeneration using animal models, stem cells, and small molecule screening with updates on preclinical developments.Inherited retinal degeneration is a group of blinding disorders afflicting more than 1 in 4000 worldwide. These disorders frequently cause the death of photoreceptor cells or retinal ganglion cells. In a subset of these disorders, photoreceptor cell death is a secondary consequence of retinal pigment epithelial cell dysfunction or degeneration. This manuscript reviews current efforts in identifying targets and developing small molecule-based therapies for these devastating neuronal degenerations, for which no cures exist. Photoreceptors and retinal ganglion cells are metabolically demanding owing to their unique structures and functional properties. Modulations of metabolic pathways, which are disrupted in most inherited retinal degenerations, serve as promising therapeutic strategies. In monogenic disorders, great insights were previously obtained regarding targets associated with the defective pathways, including phototransduction, visual cycle, and mitophagy. In addition to these target-based drug discoveries, we will discuss how phenotypic screening can be harnessed to discover beneficial molecules without prior knowledge of their mechanisms of action. Because of major anatomical and biological differences, it has frequently been challenging to model human inherited retinal degeneration conditions using small animals such as rodents. Recent advances in stem cell-based techniques are opening new avenues to obtain pure populations of human retinal ganglion cells and retinal organoids with photoreceptor cells. We will discuss concurrent ideas of utilizing stem-cell-based disease models for drug discovery and preclinical development.

Biomimetic cardiac tissue culture model (CTCM) to emulate cardiac physiology and pathophysiology ex vivo

There is need for a reliable in vitro system that can accurately replicate the cardiac physiological environment for drug testing. The limited availability of human heart tissue culture systems has led to inaccurate interpretations of cardiac-related drug effects. Here, we developed a cardiac tissue culture model (CTCM) that can electro-mechanically stimulate heart slices with physiological stretches in systole and diastole during the cardiac cycle. After 12 days in culture, this approach partially improved the viability of heart slices but did not completely maintain their structural integrity. Therefore, following small molecule screening, we found that the incorporation of 100 nM tri-iodothyronine (T3) and 1 μM dexamethasone (Dex) into our culture media preserved the microscopic structure of the slices for 12 days. When combined with T3/Dex treatment, the CTCM system maintained the transcriptional profile, viability, metabolic activity, and structural integrity for 12 days at the same levels as the fresh heart tissue. Furthermore, overstretching the cardiac tissue induced cardiac hypertrophic signaling in culture, which provides a proof of concept for the ability of the CTCM to emulate cardiac stretch-induced hypertrophic conditions. In conclusion, CTCM can emulate cardiac physiology and pathophysiology in culture for an extended time, thereby enabling reliable drug screening.

A novel high-throughput screening strategy for targeting alpha-synuclein and other long-lived proteins.

Small-molecule high-throughput screening (HTS) campaigns have frequently been used to identify lead molecules that can alter expression of disease-relevant proteins in cell-based assays. However, most cell-based HTS assays require short compound exposure periods to avoid toxicity and ensure that compounds are stable in media for the duration of the exposure. This limits the ability of HTS assays to detect inhibitors of the synthesis of target proteins with long half-lives, which can often exceed the exposure times utilized in most HTS campaigns. One such target is alpha-synuclein (α-syn)-a protein well-known for its pathological aggregation in Parkinson's Disease (PD) and other forms of neurodegeneration known collectively as synucleinopathies. Here, we report the development of an HTS assay using a CRISPR-engineered neuroblastoma cell line expressing a destabilized luciferase reporter inserted at the end of the coding region of the SNCA locus. The resultant destabilized fusion protein exhibited a significant reduction in half-life compared to the endogenous, unmodified α-syn protein, and accurately reported reductions in α-syn levels due to known protein translation inhibitors and specific α-syn siRNAs. The robustness and utility of this approach was shown by using the resulting cell line (dsLuc-Syn) to screen a focused library of 3,192 compounds for reduction of α-syn. These data demonstrate the general utility of converting endogenous loci into destabilized reporter genes capable of identifying inhibitors of gene expression of highly stable proteins even in short-term assays.

Algorithms and Databases: Unlocking Non-Targeted Screening of Small Molecules with Ambient Ionization Mass Spectrometry

Almost all sectors of analytical chemistry are finding applications for ambient ionization mass spectrometry (AI–MS) because of its ease of use, speed of analysis, and sensitivity. Although emphasis has been placed on developing new hardware that can help analyze unique samples across various applications, there has not been much innovation in the functionality of software tools and mass spectral libraries to support applications like non-targeted searching. In this article, we discuss new algorithms and libraries that have enabled non-targeted analysis of small molecules using AI–MS, as well as some of the key considerations and outstanding questions in the field.

Use of Vitex doniana (black plum) and Abutilon hirtum (Florida keys) extracts as an integral part of phytomedicine in tackling multidrug-resistant Salmonella.

The high prevalence and global spread of antibiotic resistance is driving the search for new antibacterial agents. Screening small molecules against specific bacterial targets has not yielded new compounds therefore functional assays and phenotypic screens are now being used. In Nigeria, drug resistance towards Salmonella is a major public health concern. Nine fully characterized clinical Salmonella isolates, from the Department of Medical Microbiology, Jos University Teaching Hospital, Plateau State, Nigeria, were screened by broth microdilution for susceptibility to fractionated ethanol extracts of Vitex doniana and Abutilon hirtum. This was compared to the control organism ATCC25922 and a range antibiotics: CH (chloramphenicol), SP (sparfloxacin), AM (amoxicillin), CN (gentamicin), S (streptomycin), PEF (pefloxacin). The most common resistance profile was AM,CN,S with most isolates susceptible to fluoroquinolones. Activity was detected from both plant extracts with MICs of extracted fractions ranging from 150 - 300 µg/mL. Interestingly both plants produced extracts with bactericidal activity from 300 - 600 µg/mL. V. doniana exhibited better activity against the resistant Salmonella strains in terms of greater inhibition zones, but A. hirtum extracts were more consistently active against all isolates. In comparison with the synthetic drugs, both plant extracts exhibited activity against more isolates - this activity was bactericidal. Nigeria needs better anti-salmonella products and these results represent a starting point for antibiotic drug discovery.

Small-molecule screening of ribonuclease L binders for RNA degradation

Small molecules targeting the ubiquitous latent ribonuclease (RNase L), which has limited sequence specificity toward single-stranded RNA substrates, hold great potential to be developed as broad-spectrum antiviral drugs by modulating the RNase L-mediated innate immune responses. The recent development of proximity-inducing bifunctional molecules, as described in the strategy of ribonuclease targeting chimeras, demonstrated that small-molecule RNase L activators can function as the essential RNase L-recruiting component to design bifunctional molecules for targeted RNA degradation. However, only a single screening study on small-molecule RNase L activators with poor potency has been reported to date. Herein, we established a FRET assay and conducted a screening of 240,000 small molecules to identify new RNase L activators with improved potency. The extremely low hit rate of less than 0.03% demonstrated the challenging nature of RNase L activation by small molecules available from current screening collections. A few hit compounds induced enhanced thermal stability of RNase L upon binding, although validation assays did not lead to the identification of compounds with significantly improved RNase L activating potency. The sulfonamide compound 17 induced a thermal shift of ~ 0.9 °C upon binding to RNase L, induced significant apoptosis in cancer cells, and showed single-digit micromolar inhibitory activity against cancer cell proliferation. This study paves the way for future structural optimization for the development of small-molecule RNase L binders.

Cryo-EM Structure and Activator Screening of Human Tryptophan Hydroxylase 2.

Human tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in the synthesis of serotonin. Its dysfunction has been implicated in various psychiatric disorders such as depression, autism, and bipolar disorder. TPH2 is typically decreased in stability and catalytic activity in patients; thus, screening of molecules capable of binding and stabilizing the structure of TPH2 in activated conformation is desired for drug development in mental disorder treatment. Here, we solved the 3.0 Å cryo-EM structure of the TPH2 tetramer. Then, based on the structure, we conducted allosteric site prediction and small-molecule activator screening to the obtained cavity. ZINC000068568685 was successfully selected as the best candidate with highest binding affinity. To better understand the driving forces and binding stability of the complex, we performed molecular dynamics simulation, which indicates that ZINC000068568685 has great potential to stabilize the folding of the TPH2 tetramer to facilitate its activity. The research might shed light on the development of novel drugs targeting TPH2 for the treatment of psychological disorders.

Fluorescence Lifetime Measurement of Prefibrillar Sickle Hemoglobin Oligomers as a Platform for Drug Discovery in Sickle Cell Disease.

The molecular origin of sickle cell disease (SCD) has been known since 1949, but treatments remain limited. We present the first high-throughput screening (HTS) platform for discovering small molecules that directly inhibit sickle hemoglobin (HbS) oligomerization and improve blood flow, potentially overcoming a long-standing bottleneck in SCD drug discovery. We show that at concentrations far below the threshold for nucleation and rapid polymerization, deoxygenated HbS forms small assemblies of multiple α2β2 tetramers. Our HTS platform leverages high-sensitivity fluorescence lifetime measurements that monitor these temporally stable prefibrillar HbS oligomers. We show that this approach is sensitive to compounds that inhibit HbS polymerization with or without modulating hemoglobin oxygen binding affinity. We also report the results of a pilot small-molecule screen in which we discovered and validated several novel inhibitors of HbS oligomerization.

Abstract P1031: A Novel Transcription Factor Combination For Direct Reprogramming To A Spontaneously Contracting Human Cardiomyocyte-Like State

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts, but has been less successful with human cells, limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations. Despite a decade of research, direct myocardial reprogramming still relies on overexpression of reprogramming factors first identified in mice: GATA4, MEF2C, TBX5. Using the Mogrify algorithm, we identified new candidate transcription factor combinations to induce cell conversion between human fibroblasts and cardiomyocytes. We developed an automated, high-throughput method for screening transcription factors, small molecules, and growth factors utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform we identified the combination of MYOCD, SMAD6, and TBX20 (MST) that can produce 40% TNNT2+MYH6+ cells in just 6 days and consistently produce spontaneous cardiomyocyte-like calcium transients and spontaneous contraction at just 25 days. These findings indicate that human cardiac direct reprogramming is feasible at similar levels to that achieved in the mouse and represents a step forward towards the possible clinical application of this direct cardiacreprogramming approach.

Pan-cancer proteomic map of 949 human cell lines.

SummaryThe proteome provides unique insights into disease biology beyond the genome and transcriptome. A lack of large proteomic datasets has restricted the identification of new cancer biomarkers. Here, proteomes of 949 cancer cell lines across 28 tissue types are analyzed by mass spectrometry. Deploying a workflow to quantify 8,498 proteins, these data capture evidence of cell-type and post-transcriptional modifications. Integrating multi-omics, drug response, and CRISPR-Cas9 gene essentiality screens with a deep learning-based pipeline reveals thousands of protein biomarkers of cancer vulnerabilities that are not significant at the transcript level. The power of the proteome to predict drug response is very similar to that of the transcriptome. Further, random downsampling to only 1,500 proteins has limited impact on predictive power, consistent with protein networks being highly connected and co-regulated. This pan-cancer proteomic map (ProCan-DepMapSanger) is a comprehensive resource available at https://cellmodelpassports.sanger.ac.uk.

Novel luciferase-based glucagon-like peptide 1 reporter assay reveals naturally occurring secretagogues.

Glucagon-like peptide 1 (GLP-1) is a hormone derived from preproglucagon. It is secreted by enteroendocrine cells in response to feeding and, in turn, acts as a critical regulator of insulin release. Modulating GLP-1 secretion holds promise as a strategy for controlling blood glucose levels. To dissect GLP-1 regulation and discover specific secretagogues, we engineered a reporter cell line introducing a luciferase within the proglucagon sequence in GLUTag cells. The assay was validated using western blotting and ELISA. A focused natural compounds library was screened. We measured luminescence, glucose uptake and ATP to investigate the mechanism by which newly found secretagogues potentiate GLP-1 secretion. The newly created reporter cell line is ideal for the rapid, sensitive and quantitative assessment of GLP-1 secretion. The small molecule screen identified non-toxic GLP-1 modulators. Quercetin is the most potent newly found GLP-1 secretagogue, while other flavonoids also potentiate GLP-1 secretion. Quercetin requires glucose and extracellular calcium to act as GLP-1 secretagogue. Our results support a mechanism whereby flavonoids cause GLUTag cells to utilize glucose more efficiently, leading to elevated ATP levels, followed by KATP channel blockade and GLP-1 exocytosis. Our methodology enabled finding of new GLP-1 secretagogues. Quercetin is a potent, naturally occurring GLP-1 secretagogue. Mechanistic studies of newly found secretagogues are possible in newly created reporter cell line. Further validation in more physiological systems, such as primary L-cells or whole organisms, is needed. GLP-1 secretagogues might serve as leads for developing alternative glucose-lowering therapies.