Search

Diacylglycerol (DAG) and phosphatidic acid (PA) are two key second messengers in signaling and metabolic pathways. Diacylglycerol kinase (DGK) phosphorylate DAG to produce PA, acting as a central switch between the various signal transduction pathways activated by these second messengers. Ten DGK isoforms (α, β, γ, δ, ε, ζ, η, θ, ι, and κ) have been identified and categorized into five classes based on their structural features. DGKα is highly expressed in several cancers including hepatoma, lymphoma and melanoma, and has been shown to promote cancer survival by positively regulating proliferative activity through several signaling pathways, attracting interest to DGKα as a potential therapeutic target. DGKα and ζ are expressed at high levels in T cells, and shown to promote T cell anergy, pointing to inhibition of these DGK isoforms as a means of enhancing T cell anti-tumor activity and restoring T cells function. Several DGK isoforms have also been implicated in disease contexts such as hypertension, autoimmunity, type II diabetes, epilepsy, and bipolar disorder. Current DGK inhibitor discovery efforts are focused on DGKα and ζ, but to our knowledge no compounds have proceeded to clinical trial-to date. To facilitate these efforts, we have developed a DGK screening panel including compound profiling assays for all DGK isoforms. This is an essential tool for identification of potent and isoform selective inhibitors towards therapeutically relevant DGK isoforms. Further, we present kinetic characterization of all isoforms and lipid substrate specificity studies of DGKs. Citation Format: Safnas F. AbdulSalam, Mia M. Eason, Holly A. Fowle, Anna L. Stuart, Kurumi Y. Horiuchi. Development of diacylglycerol kinase assays to facilitate isoform specific inhibitor discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 170.

There are currently no effective chemotherapeutic drugs approved for the treatment of diffuse intrinsic pontine glioma (DIPG), an aggressive pediatric cancer resident in the pons region of the brainstem. Radiation therapy is beneficial but not curative, with the condition being uniformly fatal. Analysis of the genomic landscape surrounding DIPG has revealed that activin receptor-like kinase-2 (ALK2) constitutes a potential target for therapeutic intervention given its dysregulation in the disease. We adopted an open science approach to develop a series of potent, selective, orally bioavailable, and brain-penetrant ALK2 inhibitors based on the lead compound LDN-214117. Modest structural changes to the C-3, C-4, and C-5 position substituents of the core pyridine ring afforded compounds M4K2009, M4K2117, and M4K2163, each with a superior potency, selectivity, and/or blood-brain barrier (BBB) penetration profile. Robust in vivo pharmacokinetic (PK) properties and tolerability mark these inhibitors as advanced preclinical compounds suitable for further development and evaluation in orthotopic models of DIPG.

The activity of the histone lysine methyltransferase NSD2 is thought to play a driving role in oncogenesis. Both overexpression of NSD2 and point mutations that increase its catalytic activity are associated with a variety of human cancers. While NSD2 is an attractive therapeutic target, no potent, selective and cell-active inhibitors have been reported to date, possibly due to the challenging nature of developing high-throughput assays for NSD2. To establish a platform for the discovery and development of selective NSD2 inhibitors, multiple assays were optimized and implemented. Quantitative high-throughput screening was performed with full-length wild-type NSD2 and a nucleosome substrate against a diverse collection of known bioactives comprising 16,251 compounds. Actives from the primary screen were further interrogated with orthogonal and counter assays, as well as activity assays with the clinically relevant NSD2 mutants E1099K and T1150A. Five confirmed inhibitors were selected for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. The identification of NSD2 inhibitors that bind the catalytic SET domain and demonstrate activity in cells validates the workflow, providing a template for identifying selective NSD2 inhibitors.

Epigenetic modifications play a crucial role in human diseases. Unlike genetic mutations, however, they do not change the underlying DNA sequences. Epigenetic phenomena have gained increased attention in the field of cancer research, with many studies indicating that they are significantly involved in tumor establishment and progression. Histone methyltransferases (HMTs) are a large group of enzymes that specifically methylate protein lysine and arginine residues, especially in histones, using S-adenosyl-L-methionine (SAM) as the methyl donor. However, in general, HMTs have no widely accepted high-throughput screening (HTS) assay format, and reference inhibitors are not available for many of the enzymes. In this study, we describe the application of a miniaturized, radioisotope-based reaction system: the HotSpot(SM) platform for methyltransferases. Since this platform employs tritiated SAM as a cofactor, it can be applied to the assay of any HMT. The key advantage of this format is that any substrate can be used, including peptides, proteins, or even nucleosomes, without the need for labeling or any other modifications. Using this platform, we have determined substrate specificities, characterized enzyme kinetics, performed compound profiling for both lysine and arginine methyltransferases, and carried out HTS for a small-library LOPAC against DOT1L. After hit confirmation and profiling, we found that suramin inhibited DOT1L, NSD2, and PRMT4 with IC₅₀ values at a low μM range.

Histone methyltransferases (HMTs) methylate either the lysine or arginine residues on histones and other proteins and play a crucial role in epigenetic regulation. Over 70 HMTs are encoded by the human genome, and many have been implicated in the aetiology of cancer, inflammatory diseases, neurodegenerative diseases and other conditions. There are currently about a dozen HMT activity assays available, and many of these assay formats are applicable to other epigenetic factors, such as histone acetyltransferases, histone deacetylases, and histone and DNA demethylases. Many factors need to be considered in selecting an HMT assay for drug discovery studies, including cost, adaptability to high-throughput screening, and rates of false positives and false negatives. This chapter describes the mechanisms of the major assay platforms available for HMT screening and profiling and presents the advantages and limitations associated with each.

Methylation of histone H3 lysine-4 (H3K4) is an important, regulatory, epigenetic post-translational modification associated with actively transcribed genes. In humans, the principal mediators of this modification are part of the MLL/SET1 family of methyltransferases, which comprises six members, MLLs1-4 and SET1A/SET1B. Aberrations in the structure, expression, and regulation of these enzymes are implicated in various disease states, making them important potential targets for drug discovery, particularly for oncology indications. The MLL/SET1 family members are most enzymatically active when part of a "core complex," the catalytic SET-domain-containing subunits bound to a subcomplex consisting of the proteins WDR5, RbBP5, Ash2L and a homodimer of DPY-30 (WRAD2). The necessity of MLL/SET1 members to bind WRAD2 for full activity is the basis of a particular drug development strategy, which seeks to disrupt the interaction between the MLL/SET1 subunits and WDR5. This strategy is not without its theoretical and practical drawbacks, some of which relate to the ease with which complexes of Escherichia coli-expressed MLL/SET1 and WRAD2 fall apart. As an alternative strategy, we explore ways to stabilize the complex, focusing on the use of an excess of WRAD2 to drive the binding equilibria toward complex formation while maintaining low concentrations of the catalytic subunits. The purpose of this approach is to seek inhibitors that bind the SET domain, an approach proven successful with the related, but inherently more stable, enhancer of zeste homolog 2 (EZH2) complex.

Microfluidic technologies offer the potential for highly productive and low-cost ultra-high throughput screening and high throughput selectivity profiling. Such technologies need to provide the flexibility of plate-based assays as well as be less expensive to operate. Presented here is a unique microarray system (the Reaction Biology [Malvern, PA] DiscoveryDot), which runs over 6,000 homogeneous reactions per 1" x 3" microarray using chemical libraries or compound dilutions printed in 1-nl volumes. A simple and rapid piezo-activation method delivers from 30 to 300 pl of biochemical targets and detector chemistries to each reaction. The fluorescent signals are detected and analyzed with conventional microarray scanners and software. The DiscoveryDot platform is highly customizable, and reduces consumption of targets and reaction chemistries by >40-fold and the consumption of compounds by >10,000-fold, compared to 384-well plate assay. We demonstrate here that the DiscoveryDot platform is compatible with conventional large-volume well-based reactions, with a Z' factor of >0.6 for many enzymes, such as the caspase family enzymes, matrix metalloproteinase, serine proteases, kinases, and histone deacetylases. The platform is well equipped for 50% inhibitory concentration (IC50) profiling studies of enzyme inhibitors, with up to 10 dilution conditions of each test compound printed in duplicate, and each microarray chip can generate over 300 IC50 measurements against a given target.

Employing phenylmalonitrile dianion chemistry, a large number of analogues of MEK inhibitor lead SH053 (IC(50)=140 nM) were rapidly synthesized leading to single digit nM inhibitors, displaying submicromolar AP-1 transcription inhibition in COS-7 cells. Compound 41, exhibiting a MEK IC(50)=12 nM showed ip activity in a TPA-induced ear edema model with an ED(50)=5 mg/kg.

Analogues of glutamyl-γ-boronate ( 1 ) were synthesized as mechanism-based inhibitors of bacterial Glu-tRNA Gln amidotransferase (Glu-AdT) and were designed to engage a putative catalytic serine nucleophile required for the glutaminase activity of the enzyme. Although 1 provides potent enzyme inhibition, structure–activity studies revealed a narrow range of tolerated chemical changes that maintained activity. Nonetheless, growth inhibition of organisms that require Glu-AdT by the most potent enzyme inhibitors appears to validate mechanism-based inhibitor design of Glu-AdT as an approach to antimicrobial development.

In search of antiinflammatory drugs with a new mechanism of action, U0126 was found to functionally antagonize AP-1 transcriptional activity via noncompetitive inhibition of the dual specificity kinase MEK with an IC50 of 0.07 microM for MEK 1 and 0.06 microM for MEK 2. U0126 can undergo isomerization and cyclization reactions to form a variety of products, both chemically and in vivo, all of which exhibit less affinity for MEK and lower inhibition of AP-1 activity than parent, U0126.

The compound U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstrate that the two compounds bind to deltaN3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for deltaN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the deltaN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.

Nonspecific protein binding is a commonly encountered problem with synthetic molecules designed as enzyme inhibitors. When the substrate for the enzymatic reaction is itself a protein, such nonspecific protein binding can also occur. Here, we demonstrate that this phenomenon can have a dramatic effect on the steady-state kinetic evaluation of such inhibitors.

A series of C-terminal deletion mutants of chicken gizzard smooth muscle caldesmon (CaD) were made using a polymerase chain reaction cloning strategy and a baculovirus expression system, and the precise locations of the functional domains of CaD involved in the regulation of actomyosin ATPase and the binding of actin, tropomyosin, and calmodulin were analyzed. Our results reveal a high affinity calmodulin-binding domain that consists of at least three calmodulin-binding determinants localized in residues 690-717, 658-689, and 628-657. The residues between positions 718 and 756 and positions 598 and 627 have no detectable calmodulin-binding site. A high affinity tropomyosin-binding domain is located between residues 718 and 756. The 159 residues at the C terminus of CaD contain multiple actin-binding determinants; the major ones are localized in the regions between residues 718 and 756 and residues 690 and 717. The amino acid residues between positions 718 and 756 contain the major determinant involved in the inhibition of the actin activation of smooth muscle myosin ATPase since CaD-(1-717) caused only 30% of the inhibition produced by the full-length CaD. Further deletion between residues 690 and 717 (CaD-(1-689) revealed a low level (10% of that seen for full-length CaD) of inhibition of the actomyosin ATPase. These data clearly demonstrate that the region of the last 66 amino acid residues at the CaD C terminus contains two or more major actin-binding motifs, one tropomyosin-binding domain, one high affinity calmodulin-binding determinant, and the domain that is responsible for the inhibition of the actin-activated ATPase of myosin.

Calponin, an actin/calmodulin-binding protein present in smooth muscle thin filaments, modulates the actin-myosin interaction and actomyosin ATPase activity of smooth muscle myosin II. Binding of myosin heads to actin under conditions that produce weak or strong binding induces conformational changes in actin. Polarized fluorimetric measurements of rhodamine-phalloidin complex and 1,5-IAEDANS specifically linked to actin in myosin-free muscle fibers (ghost fibers) and to Cys-707 in myosin head, respectively, revealed conformational changes, as determined from the changes in orientation and mobility of fluorescent probes, upon addition of calponin to ghost fibers. The effect of calponin on conformational changes produced upon binding of phosphorylated or dephosphorylated heavy meromyosin (HMM) was also determined. Subfragment-1 preparation modified with NEM (NEM-S1) or pPDM (pPDM-S1) were used as models of strong and weak binding, respectively. Calponin changed both the orientation of fluorophores on the actin and the flexibility of the actin filaments, as determined from the angle between an actin filament and the fiber axis. Changes in the flexibility of actin filaments and the orientation of fluorophores produced by phosphorylated smooth muscle HMM were similar to those seen with NEM-S1, which formed a strong-binding association with actin and caused the transition of actin monomers to the "on" state; calponin markedly inhibited this effect. In contrast, pPDM-S1 and dephosphorylated HMM induced weak binding and the transition of actin monomers to the "of" state, and these effects were enhanced by calponin. Furthermore, calponin decreased the velocity of actin filament movement over skeletal muscle myosin O gamma phosphorylated smooth muscle myosin heads in an in vitro motility assay. These results suggest that calponin induces modulation of smooth muscle contraction by inhibiting the force-producing (strong-binding) state of cross-bridges and involves changes in actin conformation.

Fetal hemoglobin, Hb F, is known to be an important factor for clinical course of sickle cell disease, as it suppresses polymerization of sickle hemoglobin. To investigate the effect of Hb F on the survival of sickle reticulocytes (young red cells) in circulation, Hb F levels in individual reticulocytes and mature erythrocytes were quantified via fluorescence image cytometry. We first examined unfractionated SS cells from 3 patients with different Hb F levels, and found that Hb F levels in reticulocyte populations were always lower than those in erythrocyte populations. This suggests that subsets of reticulocytes with lower Hb F levels are removed during maturation while those with higher Hb F levels tend to survive to become erythrocytes. The distribution of Hb F in reticulocytes was different among these patients and seems to strongly affect the survival of F-reticulocytes. We also analyzed density-separated fractions, and found that Hb F levels in reticulocytes found in the densest fraction were lower than those in lighter fractions. This suggests that reticulocytes with lower Hb F levels are susceptible to quick dehydration within their maturation period (1-2 days) in circulation.

Sickling of reticulocytes and mature erythrocytes from patients with sickle cell disease was compared at venous and arterial oxygen tensions (PO2 = 30 and 90 mm Hg). Reticulocyte-rich fractions (d < 1.06) were partially deoxygenated in two ways after incubation at PO2 = 180 mm Hg: (1) PO2 of 30 mm Hg for 1 hour, and reoxygenation to 90 mm Hg for 2 hours and (2) PO2 of 90 mm Hg and kept for 2 hours. Percentages of sickled cells were always higher (3 - 5 times) in reticulocytes than in mature erythrocytes. Percentage of sickled reticulocytes measured at PO2 = 90 mm Hg after partial deoxygenation to PO2 = 30 mm Hg was 2 times higher than that obtained directly at PO2 = 90 mm Hg. In contrast, there was no difference in percentage of sickled cells in the mature erythrocyte population under the two experimental conditions. These results suggest that reticulocytes are more susceptible to sickling under venous oxygen tension and less likely than mature erythrocytes to resume discoidal shape even at arterial oxygen tension.

A single-headed monomeric myosin (myosin I) was isolated from pig urinary bladder smooth muscles and purified to homogeneity. Myosin I from smooth muscle is composed of a 110-kDa heavy chain and three 17-kDa light chains. The heavy chain from smooth muscle myosin I does not cross-react with the antibody against conventional myosin (myosin II) from smooth muscle, but it does show antigenic similarity to adrenal medulla myosin I heavy chain. The light chain from smooth muscle myosin I is similar to calmodulin in molecular weight, amino acid composition, and migration on SDS-polyacrylamide gel electrophoresis in the presence of Ca2+. The high salt ATPase activity of myosin I in the presence of CaCl2 is higher than that in K(+)-EDTA. Smooth muscle actin causes a 5-10-fold activation of the Mg-ATPase activity of myosin I. In the presence of Ca2+, exogenous calmodulin enhances the actin-activated ATPase activity of myosin I, and the increased activity is associated with the binding of exogenous calmodulin to myosin I heavy chain. A maximum of 4 mol of light chains/mol of myosin I heavy chain is observed in the presence of exogenous calmodulin. Caldesmon, a calmodulin/actin-binding protein, inhibits the actin-activated ATPase activity of myosin I. This inhibition is reversed by exogenous calmodulin in the presence of Ca2+. The actin activation of myosin I ATPase exhibits around 50% Ca2+ sensitivity in the presence of exogenous calmodulin. When caldesmon is bound to actin, Ca2+ sensitivity is increased to 80% in the presence of calmodulin. Therefore, smooth muscle caldesmon, which is thought to play a role in the regulation of actin activation of myosin II, also regulates the actin activation of myosin I ATPase in smooth muscle.

Enzymes, the catalytic proteins, are playing pivotal roles in regulating basic cell functions. Drugs that inhibit enzyme activities cover varying aspects of diseases and offer potential cures. One of the major technologies used in the drug discovery industry for finding the enzyme inhibitors is high-throughput screening, which is facing a daunting challenge due to the fast-growing numbers of drug targets arising from genomic and proteomic research and the large chemical libraries generated from high-throughput synthesis. Chemical microarray, as a new technology, could be an excellent alternative for traditional well-based screening, since the technology can screen more compounds against more targets in parallel with a minimum amount of materials, reducing cost and increasing productivity. In this chapter, we have introduced the basic techniques and applications of chemical microarrays, and how to use them routinely for identifying enzyme inhibitors with functional-based assays. Sample assays for kinases, proteases, histone deacetylases, and phosphatases are demonstrated.

Fluorescence-based biochemical assays are sensitive and convenient to use; therefore, they are widely employed for enzyme assays and molecular interaction studies. However, when this method is applied for screening of a compound library for drug discovery, high fluorescence compounds, which usually exist in large numbers in chemical libraries, are problematic. Fluorescence Polarization (FP) and Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assays are less affected by compound fluorescence and suitable for large-scale high-throughput screening (HTS). In this section, we describe homogenous FP and TR-FRET methods for PI3-kinase (PI3K), a family of lipid kinases that is "difficult-to-do-HTS" since traditional radioisotope assays are hard to apply to HTS format. The application of FP and TR-FRET techniques for PI3K HTS will be described and advantages and disadvantages of these assays will be discussed.

Fluorescence-based biochemical assays are sensitive and convenient to use; therefore, they are widely employed for enzyme assays and molecular interaction studies. However, when this method is applied for screening of a compound library for drug discovery, high fluorescence compounds, which usually exist in large numbers in chemical libraries, are problematic. Fluorescence Polarization (FP) and Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assays are less affected by compound fluorescence and suitable for large-scale high-throughput screening (HTS). In this section, we describe homogenous FP and TR-FRET methods for PI3-kinase (PI3K), a family of lipid kinases that is "difficult-to-do-HTS" since traditional radioisotope assays are hard to apply to HTS format. The application of FP and TR-FRET techniques for PI3K HTS will be described and advantages and disadvantages of these assays will be discussed.

Enzymes, the catalytic proteins, are playing pivotal roles in regulating basic cell functions. Drugs that inhibit enzyme activities cover varying aspects of diseases and offer potential cures. One of the major technologies used in the drug discovery industry for finding the enzyme inhibitors is high-throughput screening, which is facing a daunting challenge due to the fast-growing numbers of drug targets arising from genomic and proteomic research and the large chemical libraries generated from high-throughput synthesis. Chemical microarray, as a new technology, could be an excellent alternative for traditional well-based screening, since the technology can screen more compounds against more targets in parallel with a minimum amount of materials, reducing cost and increasing productivity. In this chapter, we have introduced the basic techniques and applications of chemical microarrays, and how to use them routinely for identifying enzyme inhibitors with functional-based assays. Sample assays for kinases, proteases, histone deacetylases, and phosphatases are demonstrated.

Small molecule kinase inhibitors are promising therapeutic agents in a number of diseases, most notably cancer. However, mutations in kinases, both intrinsic and acquired, can drastically alter inhibitor sensitivity. To identify inhibitors of disease-associated mutant kinases, we conducted an unbiased functional screen of 182 small molecule kinase inhibitors against 76 mutated recombinant kinases arising from 21 cognate wild-type kinases. The results revealed novel lead compounds that were exquisitely selective for mutant kinases, including several that exhibited preferred inhibition of mutant kinases over their cognate wild-type kinases. This study provides a resource for the development of novel small molecule inhibitors against disease-associated mutant kinases and illustrates the potential of unbiased large-scale profiling as an approach to compound-centric kinase inhibitor discovery. Citation Format: Krisna C. Duong-Ly, Karthik Devarajan, Shuguang Liang, Kurumi Horiuchi, Yuren Wang, Haiching Ma, Jeffrey R. Peterson. Broad profiling reveals opportunities for selective inhibition of disease-associated mutant kinases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3649. doi:10.1158/1538-7445.AM2015-3649

DiscoveryDotTM is a novel solution-phase technology for chemical compound microarrays which has been validated for several targets (e.g. serine proteases, cysteine proteases, metalloproteinases, histone deacetylases, phosphatases and various kinases) of significance for drug discovery. The historical context of microarrays and the advantages of the DiscoveryDotTM technology are highlighted. The success of this chemical microarray technology will provide unprecedented possibility and capability for parallel functional analysis of hundreds of thousands of chemical compounds.

A central challenge in chemical biology is profiling the activity of a large number of chemical structures against hundreds of biological targets, such as kinases. Conventional 32P-incorporation or immunoassay of phosphorylated residues produces high-quality signals for monitoring kinase reactions but is difficult to use in high-throughput screening (HTS) because of cost and the need for well-plate washing. The authors report a method for densely archiving compounds in nanodroplets on peptide or protein substrate-coated microarrays for subsequent profiling by aerosol deposition of kinases. Each microarray contains over 6000 reaction centers (1.0 nL each) whose phosphorylation progress can be detected by immunofluorescence. For p60c-src, the microarray produced a signal-to-background ratio of 36.3 and Z' factor of 0.63 for HTS and accurate enzyme kinetic parameters (KmATP = 3.3 microM) and IC50 values for staurosporine (210 nM) and PP2 (326 nM) at 10 microM adenosine triphosphate (ATP). Similarly, B-Raf phosphorylation of MEK-coated microarrays was inhibited in the nanoliter reactions by GW5074 at the expected IC50 of 9 nM. Common kinase inhibitors were printed on microarrays, and their inhibitory activities were systematically profiled against B-Raf (V599E), KDR, Met, Flt-3 (D835Y), Lyn, EGFR, PDGFRbeta, and Tie2. All results indicate that this platform is well suited for kinetic analysis, HTS, large-scale IC50 determinations, and selectivity profiling.

Enzymatic formation of glutamate is critical to numerous biological pathways. However, current methods for assaying the activities of glutamate-forming enzymes are not particularly suitable for high-throughput screening in drug discovery. We present a continuous-read, fluorometric assay for high-throughput analysis of glutaminases. This assay is adapted to a microplate format and employs glutamate oxidase and horseradish peroxidase to couple glutamate formation to production of the fluorescent reporter molecule, resorufin, for enhancement of sensitivity (M. Zhou, Z. Diwu, N. Panchuk-Voloshina, and R. P. Haughland, 1997, Anal. Biochem. 253, 162–168). Described herein is the selection of suitable levels of coupling enzymes for optimal kinetic response and lag time of the reporter system, based on the kinetic characteristics of the individual coupling enzymes. Finally, implementation of the assay in a format for high-throughput kinetic analysis of glutaminases is demonstrated for Escherichia coli carbamoyl phosphate synthase. Derived kinetic constants are comparable to literature values determined using a variety of assay techniques.