High-throughput Screening Of Inhibitors Research Articles

Stepwise high-throughput virtual screening of Rho kinase inhibitors from natural product library and potential therapeutics for pulmonary hypertension

Context: Pulmonary hypertension (PH) is a devastating disease characterized by progressive elevation of pulmonary arterial pressure and vascular resistance due to pulmonary vasoconstriction and vessel remodeling. The activation of RhoA/Rho-kinase (ROCK) pathway plays a central role in the pathologic progression of PH and thus the Rho kinase, an essential effector of the ROCK pathway, is considered as a potential therapeutic target to attenuate PH.Objective: In the current study, a synthetic pipeline is used to discover new potent Rho inhibitors from various natural products.Materials and methods: In the pipeline, the stepwise high-throughput virtual screening, quantitative structure–activity relationship (QSAR)-based rescoring, and kinase assay were integrated. The screening was performed against a structurally diverse, drug-like natural product library, from which six identified compounds were tested to determine their inhibitory potencies agonist Rho by using a standard kinase assay protocol.Results: With this scheme, we successfully identified two potent Rho inhibitors, namely phloretin and baicalein, with activity values of IC50 = 0.22 and 0.95 μM, respectively.Discussion and conclusion: Structural examination suggested that complicated networks of non-bonded interactions such as hydrogen bonding, hydrophobic forces, and van der Waals contacts across the complex interfaces of Rho kinase are formed with the screened compounds.

Development and application of a fluorescent glucose uptake assay for the high-throughput screening of non-glycoside SGLT2 inhibitors

Sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors are of current interest as a treatment for type 2 diabetes. Efforts have been made to discover phlorizin-related glycosides with good SGLT2 inhibitory activity. To increase structural diversity and better understand the role of non-glycoside SGLT2 inhibitors on glycemic control, we initiated a research program to identify non-glycoside hits from high-throughput screening. Here, we report the development of a novel, fluorogenic probe-based glucose uptake system based on a Cu(I)-catalyzed [3+2] cycloaddition. The safer processes and cheaper substances made the developed assay our first priority for large-scale primary screening as compared to the well-known [14C]-labeled α-methyl-d-glucopyranoside ([14C]-AMG) radioactive assay. This effort culminated in the identification of a benzimidazole, non-glycoside SGLT2 hit with an EC50 value of 0.62μM by high-throughput screening of 41,000 compounds.

Rationally designed fluorogenic protease reporter visualizes spatiotemporal dynamics of apoptosis in vivo

Fluorescence resonance energy transfer-based reporters have been widely used in imaging cell signaling; however, their in vivo application has been handicapped because of poor signal. Although fluorogenic reporters overcome this problem, no such reporter of proteases has been demonstrated for in vivo imaging. Now we have redesigned an infrared fluorescent protein so that its chromophore incorporation is regulated by protease activity. Upon protease activation, the infrared fluorogenic protease reporter becomes fluorescent with no requirement of exogenous cofactor. To demonstrate biological applications, we have designed an infrared fluorogenic executioner-caspase reporter, which reveals spatiotemporal coordination between cell apoptosis and embryonic morphogenesis, as well as dynamics of apoptosis during tumorigenesis in Drosophila. The designed scaffold may be used to engineer reporters of other proteases with specific cleavage sequence.

Quantification of Histone H3 Lys27 Trimethylation (H3K27me3) by High-Throughput Microscopy Enables Cellular Large-Scale Screening for Small-Molecule EZH2 Inhibitors

EZH2 inhibition can decrease global histone H3 lysine 27 trimethylation (H3K27me3) and thereby reactivates silenced tumor suppressor genes. Inhibition of EZH2 is regarded as an option for therapeutic cancer intervention. To identify novel small-molecule (SMOL) inhibitors of EZH2 in drug discovery, trustworthy cellular assays amenable for phenotypic high-throughput screening (HTS) are crucial. We describe a reliable approach that quantifies changes in global levels of histone modification marks using high-content analysis (HCA). The approach was validated in different cell lines by using small interfering RNA and SMOL inhibitors. By automation and miniaturization from a 384-well to 1536-well plate, we demonstrated its utility in conducting phenotypic HTS campaigns and assessing structure-activity relationships (SAR). This assay enables screening of SMOL EZH2 inhibitors and can advance the mechanistic understanding of H3K27me3 suppression, which is crucial with regard to epigenetic therapy. We observed that a decrease in global H3K27me3, induced by EZH2 inhibition, comprises two distinct mechanisms: (1) inhibition of de novo DNA methylation and (II) inhibition of dynamic, replication-independent H3K27me3 turnover. This report describes an HCA assay for primary HTS to identify, profile, and optimize cellular active SMOL inhibitors targeting histone methyltransferases, which could benefit epigenetic drug discovery.

Highlights of This Issue

The transmembrane receptor Notch3 may act as a tumor suppressor or as an oncogene in human cancers, depending upon the cellular context. To elucidate its role in medullary thyroid cancer (MTC), Jaskula-Sztul and colleagues created a gain-of-function model in a MTC cell line containing a doxycycline-inducible Notch3 intracellular domain and validated the function of Notch3 overexpression by exposure to a novel class I HDAC inhibitor, AB3. The forced expression of Notch3 in MTC altered malignant phenotype by triggering apoptosis and reducing neuroendocrine tumor markers. These studies document the tumor suppressor role of Notch3 in MTC and propose it as a potential therapeutic target.Double-strand break (DSB) repair pathways are emerging as a clinically relevant target for cancer therapy. However, previous drug screening campaigns have focused on single target proteins with in vitro assays. In this issue, Goglia and colleagues describe a cell-based, high-throughput small molecule screen for novel DSB repair inhibitors. They utilized a unique assay permitting the simultaneous analysis of two key DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The authors report numerous novel DSB repair inhibitors, many of which were FDA-approved compounds. One of their hits was subsequently translated into a Phase I trial as a glioma radiosensitizer.Vascular endothelial growth factor (VEGF) is critical for physiologic and pathological angiogenesis, particularly in cancer. To date, several antiangiogenic therapies neutralizing VEGF have been successfully developed for clinical use. Here, Lee and colleagues created a novel glycosylated VEGF decoy receptor, called VEGF-Grab. VEGF-Grab contains VEGFR1 D2-D3 domain, the positively charged patches of which are engineered to reduce the isoelectric point. VEGF-Grab exhibited more potent decoy activity against VEGF-A and placental growth factor (PlGF), and had prolonged pharmacokinetic profiles. These advancements lead to stronger and more durable antiangiogenic and antitumor efficacy, suggesting that VEGF-Grab is a promising therapeutic candidate for antiangiogenic cancer therapy.A complex network of hyaluronan (HA) and proteoglycans in a tumor microenvironment with high levels of HA (HAhigh) forms a physical barrier capable of inhibiting access of therapeutic monoclonal antibodies (mAbs) and immune cells to tumor cells, resulting in a more aggressive tumor phenotype in several solid tumor types. HA depletion by PEGPH20 removes this barrier, which allows increased access of mAb and immune cells to the tumor cell, resulting in efficient ADCC-dependent tumor cell killing. Microscopic live cell imaging shows that a physical barrier in HAhigh tumor cells restricts live human NK cells from accessing the tumor cells.

A practical fluorogenic substrate for high-throughput screening of glutathione S-transferase inhibitors.

We report a new fluorogenic substrate for glutathione S-transferase (GST), 3,4-DNADCF, enabling the assay with a low level of nonenzymatic background reaction. Inhibitors against Noppera-bo/GSTe14 from Drosophila melanogaster were identified by high throughput screening using 3,4-DNADCF, demonstrating the utility of this substrate.

Bacteria microarrays as sensitive tools for exploring pathogen surface epitopes and recognition by host receptors

We have developed a readily adaptable microarray technology for high-throughput screening of pathogen-binding biomolecules and inhibitors of pathogen–counter-receptor interactions, based on the generation of bacteria microarrays.

Novel Direct Assay for Acetyl-CoA:α-Glucosaminide N-Acetyltransferase Using BODIPY-Glucosamine as a Substrate.

Heparan sulfate acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT) catalyzes the transmembrane acetylation of heparan sulfate in lysosomes required for its further catabolism. Inherited deficiency of HGSNAT in humans results in lysosomal storage of heparan sulfate and causes severe neurodegenerative disease, mucopolysaccharidosis III type C (MPS IIIC). MPS IIIC patients can potentially benefit from a therapeutic approach based on active site-specific inhibitors of HGSNAT used as pharmacological chaperons to modify the folding of the mutant protein in the patient's cells. This research however was hampered by the absence of the assay suitable for high-throughput screening of drug libraries for HGSNAT inhibitors. The existing method utilizing 4-methylumbelliferyl-β-D-glucosaminide (MU-βGlcN) requires the sequential action of two enzymes, HGSNAT and β-hexosaminidase, whereas the radioactive assay with [C14]-AcCoA is complicated and expensive. We describe a novel direct method to assay HGSNAT enzymatic activity using fluorescent BODIPY-glucosamine as a substrate. The specificity of the assay was tested using cultured fibroblasts of MPS IIIC patients, which showed a profound deficiency of HGSNAT activity as compared to normal controls as well as to MPS IIIA and D patients known to have normal HGSNAT activity. Known competitive HGSNAT inhibitor, glucosamine, had similar inhibition constants for MU-βGlcN and BODIPY-glucosamine acetylation reactions. Altogether our data show that novel HGSNAT assay is specific and potentially applicable for the biochemical diagnosis of MPS IIIC and high-throughput screening for HGSNAT inhibitors.

Novel 3'-Processing Integrase Activity Assay by Real-Time PCR for Screening and Identification of HIV-1 Integrase Inhibitors.

The 3′-end processing (3′P) of each viral long terminal repeat (LTR) during human immunodeficiency virus type-1 (HIV-1) integration is a vital step in the HIV life cycle. Blocking the 3′P using 3′P inhibitor has recently become an attractive strategy for HIV-1 therapeutic intervention. Recently, we have developed a novel real-time PCR based assay for the detection of 3′P activity in vitro. The methodology usually involves biotinylated HIV-1 LTR, HIV-1 integrase (IN), and specific primers and probe. In this novel assay, we designed the HIV-1 LTR substrate based on a sequence with a homology to HIV-1 LTR labeled at its 3′ end with biotin on the sense strand. Two nucleotides at the 3′ end were subsequently removed by IN activity. Only two nucleotides labeled biotin were captured on an avidin-coated tube; therefore, inhibiting the binding of primers and probe results in late signals in the real-time PCR. This novel assay has successfully detected both the 3′P activity of HIV-1 IN and the anti-IN activity by Raltegravir and sodium azide agent. This real-time PCR assay has been shown to be effective and inexpensive for a high-throughput screening of novel IN inhibitors.

A fluorescence-based screening assay for identification of hepatitis C virus NS3 helicase inhibitors and characterization of their inhibitory mechanism.

Hepatitis C virus (HCV) can establish a chronic infection in the majority of individuals infected, resulting in liver cirrhosis and hepatocellular carcinoma. Because the current standard treatment for HCV infection has limitations in terms of severe side effects, the emergence of drug resistance, and drug-drug interactions, it is desirable to develop novel antivirals that target viral proteins involved in viral replication. HCV nonstructural protein 3 (NS3) helicase, which unwinds double-stranded nucleic acids to yield single-stranded nucleic acids, is one possible target for new drug development, because it plays an essential role in viral replication. In this chapter, we describe a helicase assay based on fluorescence resonance energy transfer (FRET) that can be used for high-throughput screening of HCV NS3 helicase inhibitors. The assay uses a double-stranded RNA (dsRNA) substrate with a fluorophore-labeled strand hybridized to a quencher-labeled strand and monitors the increase in fluorescence intensity resulting from helicase-catalyzed unwinding of the dsRNA substrate. We further describe radioactive assays to directly visualize RNA strands unwound by helicase and to evaluate the ATPase and RNA-binding activities of NS3, which are linked to helicase activity, for characterization of the inhibitory mechanism.

Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.

Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σ(E) is critical for maintenance of the cell envelope. Because σ(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σ(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a σ(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σ(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σ(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σ(E), inhibits RNA polymerase holoenzyme formation, and inhibits σ(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σ(E) pathway.

A simple semi-quantitative in vivo method using H₂S detection to monitor sulfide metabolizing enzymes.

Here we present a simple in vivo microtiter plate assay using lead acetate [Pb(OAc)2]-soaked filter paper to detect H2S released by Escherichia coli metabolizing cysteine. The released H2S precipitates as brown lead sulfide (PbS) on Pb(OAc)2 soaked filter paper. The PbS stain quantitated by ImageJ software is proportional to the amount of H2S released from the culture. Expression of recombinant Acidithiobacillus ferrooxidans sulfide:quinone oxidoreductase (SQR) converts the H2S to sulfur, resulting in less PbS formation. The in vivo H2S oxidation activity of SQR was calculated based on the density of the PbS stain formed by E. coli expressing SQR compared with cells harboring the empty vector pLM1. The results are consistent with the in vitro activity of SQR measured by decylubiquinone (DUQ) reduction. This assay can be applied to sulfide metabolizing enzymatic studies, mutant screening and high-throughput inhibitor screens.

Abstract 697: Development of spheroid based high-throughput screening of cell-cell adhesion inhibitors to reverse acquired multicellular resistance

Abstract Background: Solid tumors are more resistant to chemo- and radio-therapy than in vitro cancer cells under conventional monolayer culture condition. This phenomenon can be explained by “multicellular resistance” (MCR). It is considered that one of approach for keeping out cancer cells from acquired MCR is inhibition of cell-cell/cell-matrix adherence. Several therapeutic approaches increasing effect of chemo- and radio-therapy by preventing MCR are proposed. However, reports of the approaches are still few. Therefore, we tried to develop a spheroid-based high-throughput screening method of cell-cell adherence inhibitor, for discovery of drug capable of reversing the acquired MCR. Method: For spheroid formation, A549 cells were cultured under three-dimensional condition using NanoCulture® Plate, which is compatible with imaging and HTS systems. After 3 days culture, A549 spheroids were treated with library compounds for 4 days. To evaluate a degree of cell-cell adherence of the spheroids, we used Hypoxia Probe, which is a phosphorescence substrate for visualizing of intra-spheroid hypoxicity. And the signals were detected using Celigo® imaging cytometer. Results: In NanoCulture® Plate, A549 cells formed a number of spheroids. Intra-spheroid hypoxicity was correlated with degree of cell-cell adherence. After screening of inhibitor of cell-cell adherence without cytotoxity, several candidates were evaluated the ability as chemosensitizing agent. Combination treatment with the hit compound and anticancer drug was more effective than each drug alone. Conclusion: We developed a novel high throughput screening method of cell-cell adherence inhibitors. And we acquired several hit compounds capable of reversing the acquired MCR. Note: This abstract was not presented at the meeting. Citation Format: Kazuya Arai, Manabu Itho, Atsushi Mizuno, Hiromi Miura. Development of spheroid based high-throughput screening of cell-cell adhesion inhibitors to reverse acquired multicellular resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 697. doi:10.1158/1538-7445.AM2014-697

Significance of glioma-associated oncogene homolog 1 (GLI1) expression in claudin-low breast cancer and crosstalk with the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway.

IntroductionThe recently identified claudin-low subtype of breast cancer is enriched for cells with stem-like and mesenchymal-like characteristics. This subtype is most often triple-negative (lacking the estrogen and progesterone receptors (ER, PR) as well as lacking epidermal growth factor 2 (HER2) amplification) and has a poor prognosis. There are few targeted treatment options available for patients with this highly aggressive type of cancer.MethodsUsing a high throughput inhibitor screen, we identified high expression of glioma-associated oncogene homolog 1 (GLI1), the effector molecule of the hedgehog (Hh) pathway, as a critical determinant of cell lines that have undergone an epithelial to mesenchymal transition (EMT).ResultsHigh GLI1 expression is a property of claudin-low cells and tumors and correlates with markers of EMT and breast cancer stem cells. Knockdown of GLI1 expression in claudin-low cell lines resulted in reduced cell viability, motility, clonogenicity, self-renewal, and reduced tumor growth of orthotopic xenografts. We observed non-canonical activation of GLI1 in claudin-low and EMT cell lines, and identified crosstalk with the NFκB pathway.ConclusionsThis work highlights the importance of GLI1 in the maintenance of characteristics of metastatic breast cancer stem cells. Remarkably, treatment with an inhibitor of the NFκB pathway reproducibly reduces GLI1 expression and protein levels. We further provide direct evidence for the binding of the NFκB subunit p65 to the GLI1 promoter in both EMT and claudin-low cell lines. Our results uncover crosstalk between NFκB and GLI1 signals and suggest that targeting these pathways may be effective against the claudin-low breast cancer subtype.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0444-4) contains supplementary material, which is available to authorized users.

Zebrafish xenotransplantation model for cancer stem-like cell study and high-throughput screening of inhibitors.

Xenotransplantation studies are important tools for studying cancer biology, especially for assaying tumor cell malignancy and providing cancer information in vivo. Cancer stem-like cells (CSCs) have been identified in many cancer types to drive tumor growth and recurrence, from "keeping" to "keep" resistant to chemotherapy and radiation therapy. In this study, we developed the xenotransplantation of CSCs derived from the leukemia and solid tumor cell lines using the zebrafish models. In adult zebrafish, we investigated that the xenografted leukemia stem cells (LSCs) from K562 cells could proliferate in vivo and keep the cancer property by re-transplantation. As for the solid tumor, these CSCs from DU145 cells (human prostate cancer) and HepG2 cells (human liver cancer) could form the tumor mass and even metastasis after xenotransplantation. In addition, the zebrafish embryos with CSC xenotransplantation could evaluate docetaxel in vivo efficiently and be available to screen the novel inhibitors by high-throughput manner. In summary, these zebrafish xenotransplantation models devote a good platform for the CSC mechanism investigation and anti-CSC inhibitor screening.

Detailed analysis and follow-up studies of a high-throughput screening for indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors

Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulator of immune responses and therefore an important therapeutic target for the treatment of diseases that involve pathological immune escape, such as cancer. Here, we describe a robust and sensitive high-throughput screen (HTS) for IDO1 inhibitors using the Prestwick Chemical Library of 1200 FDA-approved drugs and the Maybridge HitFinder Collection of 14,000 small molecules. Of the 60 hits selected for follow-up studies, 14 displayed IC50 values below 20 μM under the secondary assay conditions, and 4 showed an activity in cellular tests. In view of the high attrition rate we used both experimental and computational techniques to identify and to characterize compounds inhibiting IDO1 through unspecific inhibition mechanisms such as chemical reactivity, redox cycling, or aggregation. One specific IDO1 inhibitor scaffold, the imidazole antifungal agents, was chosen for rational structure-based lead optimization, which led to more soluble and smaller compounds with micromolar activity.

Assay Development and High-Throughput Screening for Inhibitors of Kaposi’s Sarcoma–Associated Herpesvirus N-Terminal Latency-Associated Nuclear Antigen Binding to Nucleosomes

Kaposi’s sarcoma–associated herpesvirus (KSHV) has a causative role in several human malignancies, especially in immunocompromised hosts. KSHV latently infects tumor cells and persists as an extrachromosomal episome (plasmid). KSHV latency-associated nuclear antigen (LANA) mediates KSHV episome persistence. LANA binds specific KSHV sequence to replicate viral DNA. In addition, LANA tethers KSHV genomes to mitotic chromosomes to efficiently segregate episomes to daughter nuclei after mitosis. N-terminal LANA (N-LANA) binds histones H2A and H2B to attach to chromosomes. Currently, there are no specific inhibitors of KSHV latent infection. To enable high-throughput screening (HTS) of inhibitors of N-LANA binding to nucleosomes, here we develop, miniaturize, and validate a fluorescence polarization (FP) assay that detects fluorophore-labeled N-LANA peptide binding to nucleosomes. We also miniaturize a counterscreen to identify DNA intercalators that nonspecifically inhibit N-LANA binding to nucleosomes, and also develop an enzyme-linked immunosorbent assay to assess N-LANA binding to nucleosomes in the absence of fluorescence. HTS of libraries containing more than 350,000 compounds identified multiple compounds that inhibited N-LANA binding to nucleosomes. No compounds survived all counterscreens, however. More complex small-molecule libraries will likely be necessary to identify specific inhibitors of N-LANA binding to histones H2A and H2B; these assays should prove useful for future screens.

Identification and Characterization of Separase Inhibitors (Sepins) for Cancer Therapy

Separase is an endopeptidase that cleaves cohesin subunit Rad21, facilitating the repair of DNA damage during interphase and the resolution of sister chromatid cohesion at anaphase. Separase activity is negatively regulated by securin and Cdk1–cyclin B in vivo. Separase overexpression is reported in a broad range of human tumors, and its overexpression in mouse models results in tumorigenesis. To elucidate further the mechanism of separase function and to test if inhibition of overexpressed separase can be used as a strategy to inhibit tumor-cell proliferation, small-molecule inhibitors of separase enzyme are essential. Here, we report a high-throughput screening for separase inhibitors (Sepins). We developed a fluorogenic separase assay using rhodamine 110–conjugated Rad21 peptide as substrate and screened a small-molecule compound library. We identified a noncompetitive inhibitor of separase called Sepin-1 that inhibits separase enzymatic activity with a half maximal inhibitory concentration (IC50) of 14.8 µM. Sepin-1 can inhibit the growth of human cancer cell lines and breast cancer xenograft tumors in mice by inhibiting cell proliferation and inducing apoptosis. The sensitivity to Sepin-1 in most cases is positively correlated to the level of separase in both cancer cell lines and tumors.

Development of a high-throughput assay for aldosterone synthase inhibitors using high-performance liquid chromatography–tandem mass spectrometry

Aldosterone plays a key role in the pathogenesis of hypertension, congestive heart failure, and chronic kidney disease. Aldosterone biosynthesis involves three membrane-bound enzymes: aldosterone synthase, adrenodoxin, and adrenodoxin reductase. Here, we report the development of a mass spectrometry-based high-throughput whole cell-based assay for aldosterone synthesis. A human adrenal carcinoma cell line (H295R) overexpressing human aldosterone synthase cDNA was established. The production of aldosterone in these cells was initiated with the addition of 11-deoxycorticosterone, the immediate substrate of aldosterone synthase. An automatic liquid handler was used to gently distribute cells uniformly to well plates. The adaption of a second automated liquid handling system to extract aldosterone from the cell culture medium into organic solvent enabled the development of 96- and 384-well plate formats for this cellular assay. A high-performance liquid chromatography–tandem mass spectrometry method was established for the detection of aldosterone. Production of aldosterone was linear with time and saturable with increasing substrate concentration. The assay was highly reproducible with an overall average Z′ value=0.49. This high-throughput assay would enable high-throughput screening for inhibitors of aldosterone biosynthesis.

Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors.

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic β-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole-cell colorimetric bioassay with a recombinant Escherichia coli expressing a plant HPPD for the herbicide analysis of β-triketones. The principle of the bioassay is based on the ability of the recombinant E. coli clone to produce a soluble melanin-like pigment, from tyrosine catabolism through p-hydroxyphenylpyruvate and homogentisate. The addition of sulcotrione, a HPPD inhibitor, decreased the pigment production. With the aim to optimize the assay, the E. coli recombinant clone was immobilized in sol-gel or agarose matrix in a 96-well microplate format. The limit of detection for mesotrione, tembotrione, sulcotrione, and leptospermone was 0.069, 0.051, 0.038, and 20μM, respectively, allowing to validate the whole-cell colorimetric bioassay as a simple and cost-effective alternative tool for laboratory use. The bioassay results from sulcotrione-spiked soil samples were confirmed with high-performance liquid chromatography.