3′,5′-Cyclic adenosine monophosphate (cAMP), the first identified second messenger, is implicated... more 3′,5′-Cyclic adenosine monophosphate (cAMP), the first identified second messenger, is implicated in diverse cellular processes involving cellular metabolism, cell proliferation and differentiation, apoptosis, and gene expression. cAMP is synthesized by adenylyl cyclase (AC), which converts ATP to cAMP upon activation of G αs-protein coupled receptors (GPCRs) in most cases and hydrolyzed by cyclic nucleotide phosphodiesterases (PDEs) to 5′-AMP. Dysregulation of cAMP signaling is implicated in a wide range of pathophysiological conditions such as cardiovascular diseases, neurodegenerative and behavioral disorders, cancers, diabetes, obesity, cataracts, and others. Therefore, cAMP targeted therapies have been and are still undergoing intense investigation for the treatment of these and other diseases. This highlights the need for developing assays to detect and monitor cAMP levels. In this study, we show cAMP Lumit assay as a highly specific homogeneous bioluminescent assay suitable for high throughput screenings with a large assay window and a wide dynamic range for cAMP detection. We believe that this assay will aid and simplify drug discovery screening efforts for cAMP signaling targeted therapies.
G Protein Coupled Receptors represent a large validated target for Drug discovery research. The G... more G Protein Coupled Receptors represent a large validated target for Drug discovery research. The Gs is coupled to activation of adenylate cyclase and Gi is coupled to inhibition of adenylate cyclase while Gq is coupled to activation of phospholipase ƒÒ. Cellular concentrations of second messengers such as cAMP and cGMP have been known to play major roles in smooth muscle relaxation, contractile activity of cardiovascular system, and central nervous system related pathologies. We report here on a new assay for determining cAMP concentrations in cellular and tissue extract and for monitoring modulation of GPCR that are linked to activation or inhibition of adenylate cyclase (Gs or Gi). The assay is homogenous, fast, HTS formatted with high Z¡¦, and adaptable to multi plate formats. We have successfully generated EC50 values for agonists and IC50 values for antagonists of Gs coupled receptors that are similar to those reported in the literature. The assay is easy to use, and can be carr...
Protein phosphatases are critical components in cellular regulation; they do not only act as anti... more Protein phosphatases are critical components in cellular regulation; they do not only act as antioncogenes by antagonizing protein kinases, but they also play a positive regulatory role in a variety of cellular processes that require dephosphorylation. Thus, assessing the function of these enzymes necessitates the need for a robust, sensitive assay that accurately measures their activities. The authors present a novel, homogeneous, and nonradioactive assay to measure the enzyme activity of low concentrations of several protein phosphatases (phosphoserine/phosphothreonine phosphatases and phosphotyrosine phosphatases). The assay is based on the use of fluorogenic peptide substrates (rhodamine 110, bis-phosphopeptide amide) that do not fluoresce in their conjugated form, which is resistant to cleavage by aminopeptidases. However, upon dephosphorylation by the phosphatase of interest, the peptides become cleavable by the protease and release the highly fluorescent-free rhodamine 110. The assay is rapid, can be completed in less than 2 h, and can be carried out in multiwell plate formats such as 96-, 384-, and 1536-well plates. The assay has an excellent dynamic range, high signal-to-noise ratio, and a Z′ of more than 0.8, and it is easily adapted to a robotic system for drug discovery programs targeting protein phosphatases.
The STimulator of INterferon Genes (STING) is a transmembrane protein located at the endoplasmic ... more The STimulator of INterferon Genes (STING) is a transmembrane protein located at the endoplasmic reticulum, which serves as a molecular hub in response to cytoplasmic cyclic dinucleotide (CDN) second messengers such as cyclic GMP-AMP (cGAMP). Upon stimulation of cyclic GMP-AMP synthase (cGAS) by a wide array of pathogens that provide extracellular microbial DNA and by intracellular (nuclear and mitochondrial) DNA, the second messenger cGAMP is generated, which then binds directly to the STING protein resulting in the activation of the STING pathway. This activation by cytosolic DNA initiates a cascade of events, cGAS-STING-TBK1-IRF3 signaling, that induces type 1 IFN response, and eventually the signaling is terminated by degradation of pathway components and clearance of stimulatory DNA. Given the significant roles of STING as an adaptor for DNA sensors to pathogens and its involvement in immune sensing, tumor growth control, and control of autoinflammatory and autoimmune disorders, the STING pathway has become a very important drug target. cGAS is well-positioned to act as a DNA sensor that triggers innate immune responses and initiates host immune responses against pathogens and cancer, but inappropriate activation of STING signaling causes severe and often fatal autoimmune or autoinflammatory diseases. Credible evidence suggests that the cGAS-cGAMP-STING pathway makes fundamental contributions to at least three major cancer therapies: radiation therapy, chemotherapy, and immunotherapy, and it thus represents a promising drug target. Therefore, there is a need to identify lead compounds that effectively modulate human STING for further drug development. Towards this goal, we have developed a bioluminescent assay to monitor the concentration of cGAMP in intracellular as well as extracellular compartments. The assay relies on the new concept of nanoluciferase (nanoluc) complementation where a small peptide (SmBiT) linked to cGAMP through a linker (cGAMP tracer) can be recognized by an anti cGAMP antibody that is linked to a nanoluc complementary fragment (LgBiT) resulting in bioluminescence upon the addition of nanoluc substrate. The presence of free cGAMP that is generated in the reaction competes with cGAMP tracer and decreases bioluminescence. The assay is sensitive and can detect less than 10 nM cGAMP and less than one ng of cGAS and thus, it is suitable for testing compounds that modulate cGAS activity. We will show data that correlate the activation of cGAS using less than 10 ng of dsDNA stimulation, and the activation of IFN response element reporter assays to detect cytokine release upon cGAS-STING activation. The assay is homogenous, HTS formatted and can be completed in less than one hour. Thus, this assay provides valuable contribution for investigating modulators of the STING pathway and facilitates the search for novel therapeutics. Citation Format: Said A. Goueli, Kevin Hsiao, Hui Wang, Matt Larsen. Detection and quantification of cellular GAMP using homogenous HTS formatted bioluminescent assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2765.
Protein B23 is a nucleolar and nuclear matrix phosphoprotein which has been implicated in ribosom... more Protein B23 is a nucleolar and nuclear matrix phosphoprotein which has been implicated in ribosomal assembly and/or rRNA processing. Since androgen action in the prostate is accompanied by early changes in rRNA synthesis, we have investigated androgenic regulation of protein B23 expression and phosphorylation in rat ventral prostatic nuclei. The mRNA for prostatic protein B23 was relatively stable and decreased only after several days of androgen deprivation. However, androgen deprivation resulted in a rapid change in the amount and phosphorylation of protein B23 in prostatic nuclei, which was reversed on administration of androgens to orchiectomized animals. Phosphorylation of protein B23 appears to be catalyzed primarily by casein kinase 2 (CK-2). Early androgenic changes in phosphorylation of protein B23 appear to relate more to modulations in the protein kinase activity than in the amount of protein B23. The androgen mediated enhancement in the amount of protein B23 and its phosphorylation precedes the cellular proliferative phase following androgen administration to castrated rats, and appear to be temporally concordant with the rRNA synthesis in the tissue. The androgen mediated changes in the amount and phosphorylation of protein B23 are specific to the prostate and are not detected in the liver nuclei. Thus, androgenic regulation of the amount and phosphorylation of prostatic protein B23 may be related to the early changes associated with androgen mediated growth of the gland.
G protein-coupled receptors (GPCRs) are involved in various physiological processes, such as beha... more G protein-coupled receptors (GPCRs) are involved in various physiological processes, such as behavior changes, mood alteration, and regulation of immune-system activity. Thus, GPCRs are popular targets in drug screening, and a well-designed assay can speed up the discovery of novel drug candidates. The Promega cAMP-Glo Assay is a homogenous bioluminescent assay to monitor changes in intracellular cyclic adenosine monophosphate (cAMP) concentrations in response to the effect of an agonist, antagonist, or test compound on GPCRs. Together with the Labcyte Echo 555 acoustic liquid handler and the Deerac Fluidics Equator HTS reagent dispenser, this setup can screen compounds in 96-, 384-, and 1536-well formats for their effects on GPCRs. Here, we describe our optimization of the cAMP-Glo assay in 1536-well format, validate the pharmacology, and assess the assay robustness for HTS. We have successfully demonstrated the use of the assay in primary screening applications of known agonist and antagonist compounds, and confirmed the primary hits via secondary screening. Implementing a high-throughput miniaturized GPCR assay as demonstrated here allows effective screening for potential drug candidates.
It is well recognized that methylation/demethylation of not only DNA, RNA, Proteins but also meth... more It is well recognized that methylation/demethylation of not only DNA, RNA, Proteins but also methylation of small molecules play major roles in modulation of the epigenome, transcriptome, neurotransmitter uptake and metabolic regulation. Recent biochemicval and biological data suggest that the activity of these enzymes and their expression level are under very strict regulation and any abnormal alteration in either one or both results in a wide varieties of pathogenic conditions such as cancer, inflammation, and neurodegenerative diseases. In addition to chromatin modulation, altered methylation of DNA and more recently mRNA have been recognized to regulate the transcriptome and the rate of message translation and stability. Furthermore, methylation of small molecules such as catechols and nicotinamide play critical roles in neurotransmitters uptake and function, and metabolic regulation, respectively. Thus, pharmacological modulation of these enzymes by small molecules will be beneficial in developing novel therapeutics for multiple unmet medical needs. Towards this goal of searching for activators/inhibitors of these enzymes for the development of next generation of drugs, screening assays for these modulators are urgently needed. To address these unmet needs, we have developed a novel assay that monitors the activities of these enzymes and their modulation by small molecules. The assay is bioluminescent based, HTS formatted and highly sensitive. A unique feature of this assay is its universality since it is based on monitoring the formation of the universal product S-adenosylhomocysteine (SAH), i.e., capable of detecting changes in activity of a broad range of methyltransferases such as DNA, RNA, protein, and small molecules. In addition, the assay has been validated for all classes of protein methyltransferases (Lysine and Arginine), and with different types of substrates (small peptides, large proteins, or even nucleosomes). This enables determining the specificity of these enzymes and their substrate requirements. The assay has high signal to background, low C.V., robust (Z’ value > 0.7), and has been validated using various plate densities such as 96-, 384, and 1536-well plates. A strong feature of this assay is its utility with broad range of substrates concentrations or the composition of the substrates (short vs. long peptides), thus enabling the generation of kinetic data and determining the mechanism of action of various modulators of methyltransferases of interest. Citation Format: Said A. Goueli, Kevin Hsiao. Monitoring small molecules and macromolecules methyltransferase activities using homogenous luminescent assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2412. doi:10.1158/1538-7445.AM2017-2412
Spontaneous degradation reactions such as oxidation, glycation, deamidation, Isomerization, and r... more Spontaneous degradation reactions such as oxidation, glycation, deamidation, Isomerization, and racemization are non-enzymatic modifications and produce functionally damaged species reflecting aging effect at the molecular level. L-asparagine (Asn) and L-aspartate (Asp) are among the most unstable residues in proteins- linked to deamidation, isomerization and racemization Rx. Under physiological conditions, succinimide-linked deamidation of Asn occurs with t1/2as short as 6 hrs while that for isoaspartate are generally 10-fold longer. A number of biological peptides and proteins possess labile Asn and Asp residues and the formation of IsoAsp at these sites adversely affect their function. The degradation of Asn and Asp sites via succinimide pathway has significant effect on protein function. Loss of function associated with isoAsp formation are found in many biologically critical proteins and is a major source of antibody instability and micro heterogeneity, Thus it is critical to accurately determine both the levels and the site of Asn deamidation in therapeutic antibodies and proteins. Although mass Spectroscopic platforms have been at the forefront of technologies to quantitate and identify sites of isoAsp formation, it fails in accurately determining Asn level and has problems with Asp isomerization due to the various steps required for sample preparation such as denaturation, reduction, alkylation, and enzyme digestion of the Ab before analysis resulting in problems with Asp isomerization and Asn determination artifacts. Although peptide mapping is the most powerful tool in isoAsp analyses, it is very time consuming and may itself actually cause sample degradation during preparation and analysis and is not always practical when large number of samples are needed for analysis. To improve on and complement existing technologies, we have developed a bioluminescent, homogenous, robust, sensitive and easy to use assay to accurately monitor the deamidation of Asn and isomerization of aspartate. The assay is based on the use of protein isoaspartate methyltransferase to catalyze the methylation of isoaspartate using S-adenosylmethionine (SAM) and resulting in the formation of S-adenosylhomocysteine and methylated isoaspartate. The assay is very sensitive, it can detect as little as 100 fmol of IsoAsp in as little as 5-10 pmol of proteins. Citation Format: Said A. Goueli, Kevin Hsiao, Rushikesh Patel, Vishal Nashine. A high through-put bioluminescent assay to monitor the deamidation of asparagine and isomerization of aspartate residues in therapeutic proteins and antibodies. [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 5434. doi:10.1158/1538-7445.AM2015-5434
Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) pro... more Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a critical mechanism for regulating protein functions affecting diverse cellular processes. Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an adenosine triphosphate (ATP)-dependent enzymatic cascade involving enzyme 1 (E1)-activating enzyme, E2-conjugating enzyme, and E3 ligase. The amount of adenosine monophosphate (AMP) produced in the first step, involving E1-mediated Ub/Ubl activation, represents an accurate measure of Ub/Ubl transfer during the process. Here we describe a novel bioluminescent assay platform, AMP-Glo, to quantify Ub/Ubl conjugation by measuring the AMP generated. The AMP-Glo assay is performed in a two-step reaction. The first step terminates the ubiquitination reaction, depletes the remaining ATP, and converts the AMP generated in the ubiquitination reaction to adenosine diphosphate (ADP), and in the second step the ADP generated is converted to ATP, which is detected as a bioluminescent signal using luciferase/luciferin, proportional to the AMP concentration and correlated with the Ub/Ubl transfer activity. We demonstrate the use of the assay to study Ub/Ubl conjugation and screen for chemical modulators of enzymes involved in the process. Because there is a sequential enhancement in light output in the presence of E1, E2, and E3, the AMP-Glo system can be used to deconvolute inhibitor specificity.
Two cAMP-independent protein kinases were purified from rat ventral-prostate and liver cytosol, a... more Two cAMP-independent protein kinases were purified from rat ventral-prostate and liver cytosol, and were designated PK-Cl and PK-C2 to distinguish them from the nuclear protein kinases described in the preceding paper. 2. The yield of the prostate enzymes was about 5% each, and about 10% each for the liver enzymes. The average fold purification of the prostatic enzymes was 1892 and 3176 for protein kinase CI and C2, respectively. Their average respective specific activity towards casein was 40,111 and 67,340 nmol 32p incorporated/hr per mg of enzyme protein. 3. protein kinase CI comprised one polypeptide of M r 39,000 which underwent phosphorylation in the presence of Mg 2+ + ATP. Protein kinase C2 comprised three polypeptides of M r 41,000; 38,000; 26,000. Of these only the Mr 26,000 polypeptide was autophosphorylated. 4. The Mg 2+ requirement for protein kinase C1 and C2 was between 1 and 4 mM depending on the nature of the protein substrate. Both enzymes were stimulated by I00 200 mM NaCl. 5. K m for ATP for C1 and C2 kinases was 0.0l mM; GTP could be used only by protein kinase C2 but with a markedly lower affinity. 6. The enzymes were active towards casein, phosvitin, dephosphophosvitin, and spermine-binding protein in vitro, but demonstrated little activity towards histones. 7. Despite several similarities in these general properties of cytosolic protein kinases Cl and C2 with those of nuclear protein kinases Nl and N2, a number of differences are also noted.
The recent pandemic outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐Cov‐2) was ... more The recent pandemic outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐Cov‐2) was first reported in Wuhan, China in late 2019 resulting in infection of over 259 million and death of over 5.17 world‐wide as of November 2021. In the USA, the number of diagnosed individuals reached 48 million with 92,000 Death cases. Despite the lower pathogenicity compared to SARS‐CoV and MERS‐CoV, the high infection rate of SARS‐CoV‐2 results in large numbers of hospitalizations and deaths worldwide.The SARS‐Cov‐2 is an enveloped virus and belongs to the beta coronavirus family containing a large and complex positive–sense single stranded RNA genome (~30kb). It binds to human ACE2 receptor with high affinity via its spike protein (S) and membrane fusion leading to the release of its genomic RNA that replicates and produce higher number of sub‐genomic RNAs. The viral RNA is then capped by nsp 14 and nsp10/16, two nonstructural proteins that catalyze the methylation of RNA to limit its degradation by cellular 5’‐3’ exonuclease and evade host immune system. These enzymes play critical role in translation of the viral RNA and its replication and thus leading to viral survival.We have used a bioluminescent high throughput enzyme assay for monitoring the activity of these enzymes in order to develop novel inhibitors which can be advanced for therapeutic treatment. We show that we can determine the activity of nanogram amounts of these enzymes and test the activity of these enzymes using different inhibitors, some of which are competitive with S‐adenosyl methionine the canonical substrate for these enzymes. We also show the effect of inhibitors that compete with the oligonucleotide substrate. Finally, we tested the effect of a peptide modulator of COVID‐2 on the activity of both nsp 10/16 and nsp 14. The assay is simple, homogenous, fast, and bioluminescent which provides a very robust platform for testing novel compounds on the viral methyltransferases leading to the generation of new drugs towards inhibiting the replication of COVID‐19.
Since the introduction of Herceptin and Rituximab in 1986 therapeutic antibodies have gained trem... more Since the introduction of Herceptin and Rituximab in 1986 therapeutic antibodies have gained tremendous momentum in treatment of broad range of several diseases such as cancer and inflammation. Selection of the clinical candidate mAb usually starts with large-scale in vitro screening and profiling of multiple mAbs to identify candidates that show high in vitro or in vivo activity, and thus it is necessarily to identify and eliminate potentially unstable mAbs during the lead selection process. Antibodies undergo a variety of degradation reactions which may result in compromised bioactivity and safety profile. The non-enzymatic post-translational modification of both deamidation of asparagine (Asn) and isomerization of aspartate (Asp) residues is one of the major chemical reactions occurring in proteins during production and storage resulting in formation of protein variants that may affect the quality, safety, and functionality of the therapeutic proteins. Current Methods (HPLC and LC/MS) for monitoring isoaspartate (isoAsp) formation are time consuming, require specialized equipment and trained personnel and are not amenable to high throughput scale (HTS). We have developed a robust, homogenous, high throughput formatted, and sensitive assay to accurately monitor the formation of isoAsp under several conditions such as new formulations, storage periods, and temperature.
3′,5′-Cyclic adenosine monophosphate (cAMP), the first identified second messenger, is implicated... more 3′,5′-Cyclic adenosine monophosphate (cAMP), the first identified second messenger, is implicated in diverse cellular processes involving cellular metabolism, cell proliferation and differentiation, apoptosis, and gene expression. cAMP is synthesized by adenylyl cyclase (AC), which converts ATP to cAMP upon activation of G αs-protein coupled receptors (GPCRs) in most cases and hydrolyzed by cyclic nucleotide phosphodiesterases (PDEs) to 5′-AMP. Dysregulation of cAMP signaling is implicated in a wide range of pathophysiological conditions such as cardiovascular diseases, neurodegenerative and behavioral disorders, cancers, diabetes, obesity, cataracts, and others. Therefore, cAMP targeted therapies have been and are still undergoing intense investigation for the treatment of these and other diseases. This highlights the need for developing assays to detect and monitor cAMP levels. In this study, we show cAMP Lumit assay as a highly specific homogeneous bioluminescent assay suitable for high throughput screenings with a large assay window and a wide dynamic range for cAMP detection. We believe that this assay will aid and simplify drug discovery screening efforts for cAMP signaling targeted therapies.
G Protein Coupled Receptors represent a large validated target for Drug discovery research. The G... more G Protein Coupled Receptors represent a large validated target for Drug discovery research. The Gs is coupled to activation of adenylate cyclase and Gi is coupled to inhibition of adenylate cyclase while Gq is coupled to activation of phospholipase ƒÒ. Cellular concentrations of second messengers such as cAMP and cGMP have been known to play major roles in smooth muscle relaxation, contractile activity of cardiovascular system, and central nervous system related pathologies. We report here on a new assay for determining cAMP concentrations in cellular and tissue extract and for monitoring modulation of GPCR that are linked to activation or inhibition of adenylate cyclase (Gs or Gi). The assay is homogenous, fast, HTS formatted with high Z¡¦, and adaptable to multi plate formats. We have successfully generated EC50 values for agonists and IC50 values for antagonists of Gs coupled receptors that are similar to those reported in the literature. The assay is easy to use, and can be carr...
Protein phosphatases are critical components in cellular regulation; they do not only act as anti... more Protein phosphatases are critical components in cellular regulation; they do not only act as antioncogenes by antagonizing protein kinases, but they also play a positive regulatory role in a variety of cellular processes that require dephosphorylation. Thus, assessing the function of these enzymes necessitates the need for a robust, sensitive assay that accurately measures their activities. The authors present a novel, homogeneous, and nonradioactive assay to measure the enzyme activity of low concentrations of several protein phosphatases (phosphoserine/phosphothreonine phosphatases and phosphotyrosine phosphatases). The assay is based on the use of fluorogenic peptide substrates (rhodamine 110, bis-phosphopeptide amide) that do not fluoresce in their conjugated form, which is resistant to cleavage by aminopeptidases. However, upon dephosphorylation by the phosphatase of interest, the peptides become cleavable by the protease and release the highly fluorescent-free rhodamine 110. The assay is rapid, can be completed in less than 2 h, and can be carried out in multiwell plate formats such as 96-, 384-, and 1536-well plates. The assay has an excellent dynamic range, high signal-to-noise ratio, and a Z′ of more than 0.8, and it is easily adapted to a robotic system for drug discovery programs targeting protein phosphatases.
The STimulator of INterferon Genes (STING) is a transmembrane protein located at the endoplasmic ... more The STimulator of INterferon Genes (STING) is a transmembrane protein located at the endoplasmic reticulum, which serves as a molecular hub in response to cytoplasmic cyclic dinucleotide (CDN) second messengers such as cyclic GMP-AMP (cGAMP). Upon stimulation of cyclic GMP-AMP synthase (cGAS) by a wide array of pathogens that provide extracellular microbial DNA and by intracellular (nuclear and mitochondrial) DNA, the second messenger cGAMP is generated, which then binds directly to the STING protein resulting in the activation of the STING pathway. This activation by cytosolic DNA initiates a cascade of events, cGAS-STING-TBK1-IRF3 signaling, that induces type 1 IFN response, and eventually the signaling is terminated by degradation of pathway components and clearance of stimulatory DNA. Given the significant roles of STING as an adaptor for DNA sensors to pathogens and its involvement in immune sensing, tumor growth control, and control of autoinflammatory and autoimmune disorders, the STING pathway has become a very important drug target. cGAS is well-positioned to act as a DNA sensor that triggers innate immune responses and initiates host immune responses against pathogens and cancer, but inappropriate activation of STING signaling causes severe and often fatal autoimmune or autoinflammatory diseases. Credible evidence suggests that the cGAS-cGAMP-STING pathway makes fundamental contributions to at least three major cancer therapies: radiation therapy, chemotherapy, and immunotherapy, and it thus represents a promising drug target. Therefore, there is a need to identify lead compounds that effectively modulate human STING for further drug development. Towards this goal, we have developed a bioluminescent assay to monitor the concentration of cGAMP in intracellular as well as extracellular compartments. The assay relies on the new concept of nanoluciferase (nanoluc) complementation where a small peptide (SmBiT) linked to cGAMP through a linker (cGAMP tracer) can be recognized by an anti cGAMP antibody that is linked to a nanoluc complementary fragment (LgBiT) resulting in bioluminescence upon the addition of nanoluc substrate. The presence of free cGAMP that is generated in the reaction competes with cGAMP tracer and decreases bioluminescence. The assay is sensitive and can detect less than 10 nM cGAMP and less than one ng of cGAS and thus, it is suitable for testing compounds that modulate cGAS activity. We will show data that correlate the activation of cGAS using less than 10 ng of dsDNA stimulation, and the activation of IFN response element reporter assays to detect cytokine release upon cGAS-STING activation. The assay is homogenous, HTS formatted and can be completed in less than one hour. Thus, this assay provides valuable contribution for investigating modulators of the STING pathway and facilitates the search for novel therapeutics. Citation Format: Said A. Goueli, Kevin Hsiao, Hui Wang, Matt Larsen. Detection and quantification of cellular GAMP using homogenous HTS formatted bioluminescent assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2765.
Protein B23 is a nucleolar and nuclear matrix phosphoprotein which has been implicated in ribosom... more Protein B23 is a nucleolar and nuclear matrix phosphoprotein which has been implicated in ribosomal assembly and/or rRNA processing. Since androgen action in the prostate is accompanied by early changes in rRNA synthesis, we have investigated androgenic regulation of protein B23 expression and phosphorylation in rat ventral prostatic nuclei. The mRNA for prostatic protein B23 was relatively stable and decreased only after several days of androgen deprivation. However, androgen deprivation resulted in a rapid change in the amount and phosphorylation of protein B23 in prostatic nuclei, which was reversed on administration of androgens to orchiectomized animals. Phosphorylation of protein B23 appears to be catalyzed primarily by casein kinase 2 (CK-2). Early androgenic changes in phosphorylation of protein B23 appear to relate more to modulations in the protein kinase activity than in the amount of protein B23. The androgen mediated enhancement in the amount of protein B23 and its phosphorylation precedes the cellular proliferative phase following androgen administration to castrated rats, and appear to be temporally concordant with the rRNA synthesis in the tissue. The androgen mediated changes in the amount and phosphorylation of protein B23 are specific to the prostate and are not detected in the liver nuclei. Thus, androgenic regulation of the amount and phosphorylation of prostatic protein B23 may be related to the early changes associated with androgen mediated growth of the gland.
G protein-coupled receptors (GPCRs) are involved in various physiological processes, such as beha... more G protein-coupled receptors (GPCRs) are involved in various physiological processes, such as behavior changes, mood alteration, and regulation of immune-system activity. Thus, GPCRs are popular targets in drug screening, and a well-designed assay can speed up the discovery of novel drug candidates. The Promega cAMP-Glo Assay is a homogenous bioluminescent assay to monitor changes in intracellular cyclic adenosine monophosphate (cAMP) concentrations in response to the effect of an agonist, antagonist, or test compound on GPCRs. Together with the Labcyte Echo 555 acoustic liquid handler and the Deerac Fluidics Equator HTS reagent dispenser, this setup can screen compounds in 96-, 384-, and 1536-well formats for their effects on GPCRs. Here, we describe our optimization of the cAMP-Glo assay in 1536-well format, validate the pharmacology, and assess the assay robustness for HTS. We have successfully demonstrated the use of the assay in primary screening applications of known agonist and antagonist compounds, and confirmed the primary hits via secondary screening. Implementing a high-throughput miniaturized GPCR assay as demonstrated here allows effective screening for potential drug candidates.
It is well recognized that methylation/demethylation of not only DNA, RNA, Proteins but also meth... more It is well recognized that methylation/demethylation of not only DNA, RNA, Proteins but also methylation of small molecules play major roles in modulation of the epigenome, transcriptome, neurotransmitter uptake and metabolic regulation. Recent biochemicval and biological data suggest that the activity of these enzymes and their expression level are under very strict regulation and any abnormal alteration in either one or both results in a wide varieties of pathogenic conditions such as cancer, inflammation, and neurodegenerative diseases. In addition to chromatin modulation, altered methylation of DNA and more recently mRNA have been recognized to regulate the transcriptome and the rate of message translation and stability. Furthermore, methylation of small molecules such as catechols and nicotinamide play critical roles in neurotransmitters uptake and function, and metabolic regulation, respectively. Thus, pharmacological modulation of these enzymes by small molecules will be beneficial in developing novel therapeutics for multiple unmet medical needs. Towards this goal of searching for activators/inhibitors of these enzymes for the development of next generation of drugs, screening assays for these modulators are urgently needed. To address these unmet needs, we have developed a novel assay that monitors the activities of these enzymes and their modulation by small molecules. The assay is bioluminescent based, HTS formatted and highly sensitive. A unique feature of this assay is its universality since it is based on monitoring the formation of the universal product S-adenosylhomocysteine (SAH), i.e., capable of detecting changes in activity of a broad range of methyltransferases such as DNA, RNA, protein, and small molecules. In addition, the assay has been validated for all classes of protein methyltransferases (Lysine and Arginine), and with different types of substrates (small peptides, large proteins, or even nucleosomes). This enables determining the specificity of these enzymes and their substrate requirements. The assay has high signal to background, low C.V., robust (Z’ value > 0.7), and has been validated using various plate densities such as 96-, 384, and 1536-well plates. A strong feature of this assay is its utility with broad range of substrates concentrations or the composition of the substrates (short vs. long peptides), thus enabling the generation of kinetic data and determining the mechanism of action of various modulators of methyltransferases of interest. Citation Format: Said A. Goueli, Kevin Hsiao. Monitoring small molecules and macromolecules methyltransferase activities using homogenous luminescent assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2412. doi:10.1158/1538-7445.AM2017-2412
Spontaneous degradation reactions such as oxidation, glycation, deamidation, Isomerization, and r... more Spontaneous degradation reactions such as oxidation, glycation, deamidation, Isomerization, and racemization are non-enzymatic modifications and produce functionally damaged species reflecting aging effect at the molecular level. L-asparagine (Asn) and L-aspartate (Asp) are among the most unstable residues in proteins- linked to deamidation, isomerization and racemization Rx. Under physiological conditions, succinimide-linked deamidation of Asn occurs with t1/2as short as 6 hrs while that for isoaspartate are generally 10-fold longer. A number of biological peptides and proteins possess labile Asn and Asp residues and the formation of IsoAsp at these sites adversely affect their function. The degradation of Asn and Asp sites via succinimide pathway has significant effect on protein function. Loss of function associated with isoAsp formation are found in many biologically critical proteins and is a major source of antibody instability and micro heterogeneity, Thus it is critical to accurately determine both the levels and the site of Asn deamidation in therapeutic antibodies and proteins. Although mass Spectroscopic platforms have been at the forefront of technologies to quantitate and identify sites of isoAsp formation, it fails in accurately determining Asn level and has problems with Asp isomerization due to the various steps required for sample preparation such as denaturation, reduction, alkylation, and enzyme digestion of the Ab before analysis resulting in problems with Asp isomerization and Asn determination artifacts. Although peptide mapping is the most powerful tool in isoAsp analyses, it is very time consuming and may itself actually cause sample degradation during preparation and analysis and is not always practical when large number of samples are needed for analysis. To improve on and complement existing technologies, we have developed a bioluminescent, homogenous, robust, sensitive and easy to use assay to accurately monitor the deamidation of Asn and isomerization of aspartate. The assay is based on the use of protein isoaspartate methyltransferase to catalyze the methylation of isoaspartate using S-adenosylmethionine (SAM) and resulting in the formation of S-adenosylhomocysteine and methylated isoaspartate. The assay is very sensitive, it can detect as little as 100 fmol of IsoAsp in as little as 5-10 pmol of proteins. Citation Format: Said A. Goueli, Kevin Hsiao, Rushikesh Patel, Vishal Nashine. A high through-put bioluminescent assay to monitor the deamidation of asparagine and isomerization of aspartate residues in therapeutic proteins and antibodies. [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 5434. doi:10.1158/1538-7445.AM2015-5434
Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) pro... more Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a critical mechanism for regulating protein functions affecting diverse cellular processes. Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an adenosine triphosphate (ATP)-dependent enzymatic cascade involving enzyme 1 (E1)-activating enzyme, E2-conjugating enzyme, and E3 ligase. The amount of adenosine monophosphate (AMP) produced in the first step, involving E1-mediated Ub/Ubl activation, represents an accurate measure of Ub/Ubl transfer during the process. Here we describe a novel bioluminescent assay platform, AMP-Glo, to quantify Ub/Ubl conjugation by measuring the AMP generated. The AMP-Glo assay is performed in a two-step reaction. The first step terminates the ubiquitination reaction, depletes the remaining ATP, and converts the AMP generated in the ubiquitination reaction to adenosine diphosphate (ADP), and in the second step the ADP generated is converted to ATP, which is detected as a bioluminescent signal using luciferase/luciferin, proportional to the AMP concentration and correlated with the Ub/Ubl transfer activity. We demonstrate the use of the assay to study Ub/Ubl conjugation and screen for chemical modulators of enzymes involved in the process. Because there is a sequential enhancement in light output in the presence of E1, E2, and E3, the AMP-Glo system can be used to deconvolute inhibitor specificity.
Two cAMP-independent protein kinases were purified from rat ventral-prostate and liver cytosol, a... more Two cAMP-independent protein kinases were purified from rat ventral-prostate and liver cytosol, and were designated PK-Cl and PK-C2 to distinguish them from the nuclear protein kinases described in the preceding paper. 2. The yield of the prostate enzymes was about 5% each, and about 10% each for the liver enzymes. The average fold purification of the prostatic enzymes was 1892 and 3176 for protein kinase CI and C2, respectively. Their average respective specific activity towards casein was 40,111 and 67,340 nmol 32p incorporated/hr per mg of enzyme protein. 3. protein kinase CI comprised one polypeptide of M r 39,000 which underwent phosphorylation in the presence of Mg 2+ + ATP. Protein kinase C2 comprised three polypeptides of M r 41,000; 38,000; 26,000. Of these only the Mr 26,000 polypeptide was autophosphorylated. 4. The Mg 2+ requirement for protein kinase C1 and C2 was between 1 and 4 mM depending on the nature of the protein substrate. Both enzymes were stimulated by I00 200 mM NaCl. 5. K m for ATP for C1 and C2 kinases was 0.0l mM; GTP could be used only by protein kinase C2 but with a markedly lower affinity. 6. The enzymes were active towards casein, phosvitin, dephosphophosvitin, and spermine-binding protein in vitro, but demonstrated little activity towards histones. 7. Despite several similarities in these general properties of cytosolic protein kinases Cl and C2 with those of nuclear protein kinases Nl and N2, a number of differences are also noted.
The recent pandemic outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐Cov‐2) was ... more The recent pandemic outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐Cov‐2) was first reported in Wuhan, China in late 2019 resulting in infection of over 259 million and death of over 5.17 world‐wide as of November 2021. In the USA, the number of diagnosed individuals reached 48 million with 92,000 Death cases. Despite the lower pathogenicity compared to SARS‐CoV and MERS‐CoV, the high infection rate of SARS‐CoV‐2 results in large numbers of hospitalizations and deaths worldwide.The SARS‐Cov‐2 is an enveloped virus and belongs to the beta coronavirus family containing a large and complex positive–sense single stranded RNA genome (~30kb). It binds to human ACE2 receptor with high affinity via its spike protein (S) and membrane fusion leading to the release of its genomic RNA that replicates and produce higher number of sub‐genomic RNAs. The viral RNA is then capped by nsp 14 and nsp10/16, two nonstructural proteins that catalyze the methylation of RNA to limit its degradation by cellular 5’‐3’ exonuclease and evade host immune system. These enzymes play critical role in translation of the viral RNA and its replication and thus leading to viral survival.We have used a bioluminescent high throughput enzyme assay for monitoring the activity of these enzymes in order to develop novel inhibitors which can be advanced for therapeutic treatment. We show that we can determine the activity of nanogram amounts of these enzymes and test the activity of these enzymes using different inhibitors, some of which are competitive with S‐adenosyl methionine the canonical substrate for these enzymes. We also show the effect of inhibitors that compete with the oligonucleotide substrate. Finally, we tested the effect of a peptide modulator of COVID‐2 on the activity of both nsp 10/16 and nsp 14. The assay is simple, homogenous, fast, and bioluminescent which provides a very robust platform for testing novel compounds on the viral methyltransferases leading to the generation of new drugs towards inhibiting the replication of COVID‐19.
Since the introduction of Herceptin and Rituximab in 1986 therapeutic antibodies have gained trem... more Since the introduction of Herceptin and Rituximab in 1986 therapeutic antibodies have gained tremendous momentum in treatment of broad range of several diseases such as cancer and inflammation. Selection of the clinical candidate mAb usually starts with large-scale in vitro screening and profiling of multiple mAbs to identify candidates that show high in vitro or in vivo activity, and thus it is necessarily to identify and eliminate potentially unstable mAbs during the lead selection process. Antibodies undergo a variety of degradation reactions which may result in compromised bioactivity and safety profile. The non-enzymatic post-translational modification of both deamidation of asparagine (Asn) and isomerization of aspartate (Asp) residues is one of the major chemical reactions occurring in proteins during production and storage resulting in formation of protein variants that may affect the quality, safety, and functionality of the therapeutic proteins. Current Methods (HPLC and LC/MS) for monitoring isoaspartate (isoAsp) formation are time consuming, require specialized equipment and trained personnel and are not amenable to high throughput scale (HTS). We have developed a robust, homogenous, high throughput formatted, and sensitive assay to accurately monitor the formation of isoAsp under several conditions such as new formulations, storage periods, and temperature.
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Papers by Said Goueli