A Sub-Micromolar MraYAA Inhibitor with an Aminoribosyl Uridine Structure and a (S,S)-Tartaric Diamide: Synthesis,Biological Evaluation and Molecular Modeling

New inhibitors of the bacterial tranferase MraY are described. Their structure is based on an aminoribosyl uridine scaffold, which is known to be important for the biological activity of natural MraY inhibitors. A decyl alkyl chain was introduced onto this scaffold through various linkers. The synthesized compounds were tested against the MraY_AA transferase activity, and the most active compound with an original (S,S)-tartaric diamide linker inhibits MraY activity with an IC₅₀ equal to 0.37 µM. Their antibacterial activity was also evaluated on a panel of Gram-positive and Gram-negative strains; however, the compounds showed no antibacterial activity. Docking and molecular dynamics studies revealed that this new linker established two stabilizing key interactions with N190 and H325, as observed for the highly potent inhibitors carbacaprazamycin, muraymycin D2 and tunicamycin.     

A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O‐GlcNAc Transferase

Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high‐throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead‐based strategy to measure the group‐transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O‐GlcNAc transferase (OGT) as a model system through detailed Michaelis–Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high‐throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group‐transfer enzymes.     

Screening for inhibitors of histone deacetylase by incorporating a spraying method to micro-arrayed compound screening

We have developed a method of spraying assay reagents onto a target gel in the Micro-Arrayed Compound Screening ( micro ARCS) format. After application of target gels to compound sheets, subsequent reagents can be applied by spraying onto the target gel. The spraying method conserves on assay reagents by up to 10-fold, eliminates the need for casting additional agarose gels, and increases the throughput of a screen by 3-fold. To demonstrate the efficacy of applying the spraying method to micro ARCS, we screened over 600,000 compounds for inhibitors of histone deacetylase (HDAC). Commercially available HDAC substrate and reaction developer were sprayed directly onto the gel to initiate the reaction and to amplify the signal, respectively. Picks in the primary screen were retested at a density of 384 compounds per sheet in the micro ARCS format. IC(50) values for active compounds were confirmed in a 96-well plate assay. The screen identified several small molecule inhibitors of the enzyme, including members of several classes of known HDAC inhibitors. The combination of the high-density format of micro ARCS, the efficiency of the spraying method, and a timed sequence of adding assay reagents permitted a screening throughput of 200,000 tests an hour. We present the details of the screening format and the analysis of the hits from the screen.     

Bioorthogonal Probes for the Study of MDM2‐p53 Inhibitors in Cells and Development of High‐Content Screening Assays for Drug Discovery

To study the behavior of MDM2‐p53 inhibitors in a disease‐relevant cellular model, we have developed and validated a set of bioorthogonal probes that can be fluorescently labeled in cells and used in high‐content screening assays. By using automated image analysis with single‐cell resolution, we could visualize the intracellular target binding of compounds by co‐localization and quantify target upregulation upon MDM2‐p53 inhibition in an osteosarcoma model. Additionally, we developed a high‐throughput assay to quantify target occupancy of non‐tagged MDM2‐p53 inhibitors by competition and to identify novel chemical matter. This approach could be expanded to other targets for lead discovery applications.     

A Small‐Molecule Protein–Protein Interaction Inhibitor of PARP1 That Targets Its BRCT Domain

Poly(ADP‐ribose)polymerase‐1 (PARP1) is a BRCT‐containing enzyme (BRCT=BRCA1 C‐terminus) mainly involved in DNA repair and damage response and a validated target for cancer treatment. Small‐molecule inhibitors that target the PARP1 catalytic domain have been actively pursued as anticancer drugs, but are potentially problematic owing to a lack of selectivity. Compounds that are capable of disrupting protein–protein interactions of PARP1 provide an alternative by inhibiting its activities with improved selectivity profiles. Herein, by establishing a high‐throughput microplate‐based assay suitable for screening potential PPI inhibitors of the PARP1 BRCT domain, we have discovered that (±)‐gossypol, a natural product with a number of known biological activities, possesses novel PARP1 inhibitory activity both in vitro and in cancer cells and presumably acts through disruption of protein–protein interactions. As the first known cell‐permeable small‐molecule PPI inhibitor of PAPR1, we further established that (?)‐gossypol was likely the causative agent of PARP1 inhibition by promoting the formation of a 1:2 compound/PARP1 complex by reversible formation of a covalent imine linkage.     

FRET-based direct and continuous monitoring of human fucosyltransferases activity: an efficient synthesis of versatile GDP-L-fucose derivatives from abundant D-galactose

We have developed a facile and versatile protocol for the continuous monitoring of human fucosyltransferases activity by using fluorescence energy resonance transfer (FRET), and have explored the feasibility of its use in an inhibitor screening assay. A convenient sugar nucleotide with a fluorogenic probe, 6-deoxy-6-N-(2-naphalene-2-yl-acetamide)-beta-L-galactopyranos-1-yl-guanosine 5'-diphosphate disodium salt (1), was efficiently synthesized from naturally abundant D-galactopyranose via a key intermediate, 6-azide-1,2,3,4-tetra-O-benzoyl-6-deoxy-beta-L-galactopyranose (10). It was demonstrated that the combined use of the glycosyl donor 1 and a dansylated acceptor substrate, sialyl-alpha2,3-LacNAc derivative (2) allowed us to carry out highly sensitive, direct, and continuous in vitro monitoring of the generation of sialyl Lewis X (SLe x), which is catalyzed by human alpha-1,3-fucosyltransferase VI (FUT-VI). A kinetic analysis revealed that compound 1 was an excellent donor substrate (KM=0.94 microM and Vmax=0.14 microM min(-1)) for detecting human FUT-VI activity. To the best of our knowledge, this synthetic fluorogenic probe is the most sensitive and selective donor substrate for FUT-VI among all of the known GDP-Fuc analogues, including the parent GDP-Fuc. When a dansylated asparagine-linked glycopeptide 20, which is derived from egg yolk was employed as an alternate acceptor substrate, a FRET-based assay with compound 1 could be used to directly monitor the alpha1,6-fucosylation at the reducing terminal GlcNAc residue by human FUT-VIII (KM=175 microM and Vmax=0.06 microM/ min); this indicates that the present method might become a general protocol for the characterization of various mammalian fucosyltransferases in the presence of designated fluorogenic acceptor substrates. The present protocol revealed that compound 23, which was obtained by a 1,3-dipolar cycloaddition between the disodium salt 16 and 1-ethynyl-naphthalene exhibits highly potent inhibitory effects against the FUT-VI-mediated sialyl Lewis X synthesis (IC50=5.4 microM).     

Extending matrix‐assisted laser desorption/ionization triple quadrupole mass spectrometry enzyme screening assays to targets with small molecule substrates

Mass spectrometry (MS)‐based high‐throughput screening (HTS) has tremendous potential as an alternative to current screening methods due to its speed, sensitivity, reproducibility and label‐free readout. We recently reported that a new generation matrix‐assisted laser desorption/ionization triple quadrupole (MALDI‐QqQ) mass spectrometer is ideally suited for a variety of enzyme assays and screening protocols. However, all the targets measured to date had peptide substrates that were easily monitored by selected ion monitoring (SIM) without interference from the MALDI matrix. To further extend the application to enzymes with small molecule, non‐peptide substrates, we evaluated this method for measuring enzyme activity and inhibition of acetylcholinesterase (AChE). Due to the potential of MALDI matrix interference, multiple reaction monitoring (MRM) was investigated for selective MS/MS transitions and to accurately measure the conversion of acetylcholine into choline. Importantly, ionization, detection and MRM transition efficiency differences between the substrate and product can be overcome by pre‐balancing the MRM transitions during method development, thus allowing for a direct readout of the enzyme activity using the ratio of the substrate and product signals. Further validation of the assay showed accurate concentration‐dependent inhibition measurements of AChE with several known inhibitors. Finally, a small library of 1008 drug‐like compounds was screened at a single dose (10 µM) and the top 10 inhibitors from this primary screen were validated in a secondary screen to determine the rank order of inhibitory potency for each compound. Collectively, these data demonstrate that a MALDI‐QqQMS‐based readout platform is amenable to measuring small molecule substrates and products and offers significant advantages over current HTS methods in terms of speed, sensitivity, reproducibility and reagent costs. Copyright © 2009 John Wiley & Sons, Ltd.     

Use of stable isotope labeled probes to facilitate liquid chromatography/mass spectrometry based high‐throughput screening of time‐dependent CYP inhibitors

Inhibition curve shift is a commonly used approach for screening of time‐dependent CYP inhibitors which requires parallel paired incubations to obtain two inhibition curves for comparison. For the control incubation, a test compound is co‐incubated with a probe substrate in human liver microsomes (HLM) fortified with NADPH; for the time‐dependent incubation (TDI), the test compound is pre‐incubated with NADPH‐fortified HLM followed by a secondary incubation with a probe substrate. For both incubations, enzyme activity is measured respectively by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of the CYP‐specific metabolite, and a TDI inhibitor can be readily identified by inhibition curve shifting as a result of CYP inactivation by the test compound during the pre‐incubation. In the present study, we describe an alternative approach to facilitate TDI screening in which stable isotope labeled CYP‐specific probes are used for the TDI, and non‐labeled substrates are included in the control incubation. Because CYP‐specific metabolites produced in the TDI are stable isotope labeled, two sets of incubation samples can be combined and then simultaneously analyzed by LC/MS/MS in the same batch run to reduce the run time. This new method has been extensively validated using both a number of known competitive and TDI inhibitors specific to five most common CYPs such as 1A2, 2C9, 2C19, 2D6, and 3A4. The assay is performed in a 96‐well format and can be fully automated. Compared to the traditional method, this approach in combination with sample pooling and a short LC/MS/MS gradient significantly enhances the throughput of TDI screening and thus can be easily implemented in drug discovery to evaluate a large number of compounds without adding additional resource. Copyright © 2010 John Wiley & Sons, Ltd.     

A high-throughput sphingomyelinase assay using natural substrate

Sphingomyelinases are a group of hydrolases that cleave sphingomyelin, a common component of plasma membranes, to form ceramide and phosphocholine. Ceramide is a second messenger that is present in virtually all cell types and regulates a variety of cellular functions such as proliferation, differentiation, apoptosis, and inflammation response. Inhibition of sphingomyelinase activity to reduce ceramide concentrations has recently emerged as a potential therapeutic approach for several diseases including atherosclerosis, pathogen infections, inflammation, diabetes, and obesity. To effectively screen compound collections for the identification of new sphingomyelinase inhibitors, we have developed a high-throughput assay utilizing the natural substrate sphingomyelin in 1,536-well plate format. The assay has a signal-to-basal ratio of 6.1-fold in pH?5.0 buffer and 4.3-fold in pH?6.5 buffer, indicating a robust assay for compound library screening. A screen of ~300,000 compounds using this assay led to the identification of eight compounds as sphingomyelinase inhibitors (IC(50)s?=?1.7 to 38.2?μM) that exhibited different activities between the natural substrate assay and profluorescence substrate assay. The results demonstrate the robustness and effectiveness of the natural substrate sphingomyelinase assay for screening sphingomyelinase inhibitors.     

Structure Elucidation of the First Inhibitors of Human Papillomavirus Type 11 E1?E2 Protein?Protein Interaction

A novel series of inhibitors of the HPV11 E1? E2 protein? protein interaction was identified. These inhibitors, which were discovered as a result of high‐throughput screening, feature an indandione system spiro‐fused onto an appropriately substituted tetrahydrofuran ring. Early stability studies indicated, surprisingly, that this particular series of compounds were readily converted, in binding assay buffer, to the corresponding carboxylates. NMR and mass spectrometry techniques were used to elucidate the structures of these products and the mechanism by which they are produced.     

A CIEF‐LIF method for simultaneous analysis of multiple protein kinases and screening of inhibitors

Here, a CIEF‐LIF method for multiple protein kinase simultaneous analysis and inhibitors throughput screening with fast rate and low cost is presented. Comparing with CZE, CIEF‐LIF exhibited great focusing ability and high separation efficiency for substrate and phosphorylated peptides, and is applicable for multiple kinases simultaneous analysis regardless of their substrate peptides compositions and charge statuses. Thus, highly sensitive analysis for cyclic adenosine 3’, 5’‐monophosphate‐dependent protein kinase (PKA) and cyclin‐dependent kinase 1 (CDK1) was achieved in CIEF‐LIF analysis with detection sensitivity up to 1.25 mU/μL and 0.4 mU/μL, respectively, two magnitudes higher than that of CZE and comparable with that in nanomaterials or green fluorescent protein‐based kinase assay. Moreover, the inhibition effect of inhibitors on multiple kinases could be simultaneously readout in a single electrophoretic run, with half maximal inhibitory concentration of H‐89 for PKA and Ro‐3306 for CDK1 calculated as 37.0 and 35.9 nM, respectively, consistent with literatures reported. The CIEF‐LIF also exhibited strong anti‐interference ability in human breast cancer cell lysates analysis and simulators such as forskolin and 3‐isobutyl‐1‐methylxantine assessment. Therefore, CIEF‐LIF is desirable for future biological application and clinical diagnostics and drug discovery.     

Capillary electrophoresis with laser-induced fluorescence detection as a tool for enzyme characterization and inhibitor screening.

An effective, rapid and economical CE/LIF (capillary electrophoresis/laser-induced fluorescence) method was developed and applied to the characterization of signal peptidase (SPase) enzyme, which is a target for the screening of new drug candidates. In this method, CE separates the product from the substrate and LIF selectively detects the fluorescence-labeled product and substrate. By measuring the increase of the product as a function of time, one can monitor the progression of the enzyme reaction. The progression curves were also used for screening inhibitors for this enzyme. The effects of various reaction conditions were also studied and discussed. In addition, this CE/LIF method was applied to the determination of the enzyme activity, the quality control of the substrate and/or enzymes, and the cross-reactivity of inhibitors to the enzyme. It can be concluded that this method is suitable for high throughput screening (HTS) assays because it can deliver fast, sensitive, quantitative, and reliable results.     

The application of multiplexed,multi‐dimensional ultra‐high‐performance liquid chromatography/tandem mass spectrometry to the high‐throughput screening of lysosomal storage disorders in newborn dried bloodspots

Lysozomal storage disorders are just beginning to be routinely screened using enzyme activity assays involving dried blood spots and tandem mass spectrometry (MS/MS). This paper discusses some of the analytical challenges associated with published assays including complex sample preparation and potential interference from excess residual substrate. Solutions to these challenges are presented in the form of on‐line two‐dimensional chromatography to eliminate off‐line liquid‐liquid extraction (LLE) and solid‐phase extraction (SPE), the use of ultra‐high‐performance liquid chromatography (UHPLC) to separate excess substrate from all other analytes and multiplexed sample introduction for higher throughput required of a population screening assay. High sensitivity, specificity and throughput were demonstrated using this novel method. Copyright © 2010 John Wiley & Sons, Ltd.     

A High‐Throughput Assay for Arylamine Halogenation Based on a Peroxidase‐Mediated Quinone–Amine Coupling with Applications in the Screening of Enzymatic Halogenations

Arylhalides are important building blocks in many fine chemicals, pharmaceuticals and agrochemicals, and there has been increasing interest in the development of more “green” halogenation methods based on enzyme catalysis. However, the screening and development of new enzymes for biohalogenation has been hampered by a lack of high‐throughput screening methods. Described herein is the development of a colorimetric assay for detecting both chemical and enzymatic arylamine halogenation reactions in an aqueous environment. The assay is based on the unique UV/Vis spectrum created by the formation of an ortho‐benzoquinone‐amine adduct, which is produced by the peroxidase‐catalysed benzoquinone generation, followed by Michael addition of either a halogenated or non‐halogenated arylamine. This assay is sensitive, rapid and amenable to high‐throughput screening platforms. We have also shown this assay to be easily coupled to a flavin‐dependent halogenase, which currently lacks any convenient colorimetric assay, in a “one‐pot” workflow.     

Stochastic DNA Walkers in Droplets for Super‐Multiplexed Bacterial Phenotype Detection

Pathogen detection is growing in importance in the global health arena because of the high morbidity and mortality associated with bacterial blood stream infections. In this work, we present stochastic DNA walkers in droplets (SDwalker‐Drop), a one‐step, rapid, and super‐multiplex method for ultrahigh‐throughput bacterial detection. The SDwalkers, by exploiting cascade signal amplification, endow our analytical platform with fast analysis times and single‐cell analysis ability. The autonomous and multiple‐step walking behavior of the SDwalkers provides a super‐multiplex droplet‐encoding strategy by embedding intensity coded barcodes into a sequence of color‐multiplexed barcodes. We realized a theoretical coding capacity of 8³?1=511 and achieved 20 distinct patterns for bacterial phenotype detection and identification. Moreover, our SDwalker‐Drop platform could be readily integrated with a flow cytometer to afford a general approach for super‐multiplexed, high‐throughput biological assays and screening.     

Design and Synthesis of New 9‐Substituted Norharmane Derivatives as Potential Sirt5 Inhibitors

Sirt5 is a potential new drug target for the treatment of cancer, Alzheimer's disease, and Parkinson's disease. Given that norharmane is an important chemical synthon for some biologically important compounds and 9‐substituted norharmane derivatives containing a negatively charged carboxyl group may accord with the characteristic of potential Sirt5 inhibitors, a series of novel 9‐substituted norharmane derivatives were synthesized. The chemical structures and purities of all the target compounds were characterized by ¹H NMR, ¹³C NMR, MS, and HPLC. By in vitro SIRT5 inhibitory assays, three compounds ( 1a , 3a , and 3b ) show over 30% inhibition ratios at concentration of 100 µM , and the most active compound 3b has 35% and 52% inhibition ratios at 30 µM and 100 µM , respectively. Docking analysis showed that compound 3b is likely to fit very well on the substrate binding site of Sirt5, and hence, we believe that compound 3b can serve as a lead compound for further efforts to develop specific Sirt5 inhibitors.     

Fluorescence resonance energy transfer assay for high-throughput screening of ADAMTS1 inhibitors

A disintegrin and metalloprotease with thrombospondin type I motifs-1 (ADAMTS1) plays a crucial role in inflammatory joint diseases and its inhibitors are potential candidates for anti-arthritis drugs. For the purposes of drug discovery, we reported the development and validation of fluorescence resonance energy transfer (FRET) assay for high-throughput screening (HTS) of the ADAMTS1 inhibitors. A FRET substrate was designed for a quantitative assay of ADAMTS1 activity and enzyme kinetics studies. The assay was developed into a 50-μL, 384-well assay format for high throughput screening of ADAMTS1 inhibitors with an overall Z' factor of 0.89. ADAMTS1 inhibitors were screened against a diverse library of 40,960 total compounds with the established HTS system. Four structurally related hits, naturally occurring compounds, kuwanon P, kuwanon X, albafuran C and mulberrofuran J, extracted from the Chinese herb Morus alba L., were identified for further investigation. The results suggest that this FRET assay is an excellent tool, not only for measurement of ADAMTS1 activity but also for discovery of novel ADAMTS1 inhibitors with HTS.     

A Quadruplex‐Based,Label‐Free,and Real‐Time Fluorescence Assay for RNase H Activity and Inhibition

We demonstrate a unique quadruplex‐based fluorescence assay for sensitive, facile, real‐time, and label‐free detection of RNase H activity and inhibition by using a G‐quadruplex formation strategy. In our approach, a RNA–DNA substrate was prepared, with the DNA strand designed as a quadruplex‐forming oligomer. Upon cleavage of the RNA strand by RNase H, the released G‐rich DNA strand folds into a quadruplex in the presence of monovalent ions and interacts with a specific G‐quadruplex binder, N‐methyl mesoporphyrin IX (NMM); this gives a dramatic increase in fluorescence and serves as a reporter of the reaction. This novel assay is simple in design, fast in operation, and is more convenient and promising than other methods. It takes less than 30 min to finish and the detection limit is much better or at least comparable to previous reports. No sophisticated experimental techniques or chemical modification for either RNA or DNA are required. The assay can be accomplished by using a common spectrophotometer and obviates possible interference with the kinetic behavior of the catalysts. Our approach offers an ideal system for high‐throughput screening of enzyme inhibitors and demonstrates that the structure of the G‐quadruplex can be used as a functional tool in specific fields in the future.     

A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide

Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening.     

MEMS microwell and microcolumn arrays: novel methods for high-throughput cell-based assays

Although the cell-based assay is becoming more popular for high throughput drug screening and the functional characterization of disease-associated genes, most researchers in these areas do not use it because it is a complex and expensive process. We wanted to create a simple method of performing an on-chip cell-based assay. To do this, we used micro-electro-mechanical systems (MEMS) to fabricate a microwell array chip comprised of a glass substrate covered with a photoresist film patterned to form multiple microwells and tested it in two reverse transfection experiments, an exogenous gene expression study and an endogenous gene knockdown study. It was used effectively in both. Then, using the same MEMS technology, we fabricated a complementary microcolumn array to be used as a drug carrier device to topically apply drugs to cells cultured in the microwell array. We tested the effectiveness of microwell-microcolumn on-chip cell-based assay by using it in experiments to identify epidermal growth factor receptor (EGFR) activity inhibitors, for which it was found to provide effective high throughput and high content functional screening. In conclusion, this new method of cell-based screening proved to be a simple and efficient method of characterizing gene function and discovering drug leads.