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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

Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a fundamental mechanism for regulating protein functions and protein stability thus affecting diverse cellular processes. Several ubiquitin-like (Ubl) molecules have been identified including SUMO, NEDD8, ATG8/15 and ISG15 which share the ubiquitin fold Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an ATP-dependent enzymatic cascade involving E1-activating enzyme, E2-conjugating enzyme and E3-ligase. To mark proteins for degradation, multiple ubiquitins are covalently attached to produce a Lys48 linked poly-ubiquitin chain. The poly-ubiquitinated proteins are recognized by the 26S proteasome system and deubiquitinated and destroyed. Substrates with ubiquitin chains linked through Lys 6, 11, 27, 29, 33 also regulate protein degradation. Substrates marked with mono-ubiquitination or Lys63 ubiquitin chains are involved in cell signaling independent of degradative functions. Many diseases are associated with dysfunction of ubiquitin signaling, with the E3 ligase class of enzymes are considered to be the most important target. However, in recent years, evidence have accumulated demonstrating the significance of E2 class of enzymes where mutations and impairment of this class lead to severe diseases states including chromosomal instability, cancer predisposition, neurological syndromes, and immunological disorders. Thus, in addition to E3, E2 group of enzymes represent important class of therapeutic targets. To address the need for developing drugs targeting these enzymes, we have developed a novel assay that can be used to screen for modulators of these enzymes by taking advantage of the first ubiquitin activating enzyme E1 which activates free Ub forming a high-energy thioester bond between E1’s active site Cys and the C-terminus of Ub, generating AMP and pyrophosphate (PPi) as products of the reaction. We monitor the activity of these enzymes by determining the concentration of AMP generated in the reactions in a luminescence-based assay. Our assay interrogate not only E1 but also E2 and E3 in a very robust and sensitive manner. The assay is homogenous, bioluminescent, and show a very high signal to background in a very selective manner for each class of the ubiquitin pathway. The assay was successfully tested for ubiquitination, SUMOlyation and Neddylation reactions and thus can be used for screening of enzymes targeting these pathways. Citation Format: Said A. Goueli, Kevin Hsiao, Subhanjan Mondal. Dissecting the ubiquitin pathway using homogenous and sensitive bioluminescent 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 5335. doi:10.1158/1538-7445.AM2017-5335

Adenine nucleotides are major determinants of the energy status of the cell and thus any modulation of their cellular concentration has significant consequences to cellular metabolism, cellular growth and cell death. ATP generating enzymes are usually involved in anabolic processes while ATP consuming enzymes are involved in catabolite processes. We have developed biochemical and cell based assays that monitor the concentrations of ATP, ADP and AMP in biochemical reactions and in cells. These universal assays can measure the activity of various enzymes with no modification of the native substrate and the ability to use diverse substrates such as proteins, peptides, lipids, sugars, etc. The AMP generating reactions such as ubiquitin ligases, aa aminoacyl t-RNA synthetases, DNA ligases, cAMP-dependent phosphodiesterases, etc. can be also monitored with high sensitivity and reproducibility. The ADP-generating (kinases and ATPases, and ATP-consuming reactions (kinases and ATPases) can be monitored as well usi...

A variety of approaches to investigate cell signalling were presented. The main goal of the speakers was to make use of signalling pathways as targets for the development of new drugs. These targets included protein kinase A anchoring, Ras-dependent transformation, growth factor receptor tyrosine kinases (trk and insulin), cytokine-mediated signalling, phospholipid lipases and immunosuppressant targeted enzymes. Each of the speakers gave a concise summary of the area involved and the rationale supporting their approaches to investigation.

The extracellular signal-regulated kinase (ERK), originally identified as a participant in mitogenic signaling, has recently been implicated in the signaling of cellular differentiation. To examine the role of the ERK/MAP kinase pathway in megakaryocytic differentiation of K562 cells, the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin on ERK activation were determined. Both TPA and bryostatin are known to activate PKC but paradoxically have opposing effects on megakaryocytic differentiation. TPA, a differentiation inducer, caused sustained activation of ERK (24 h), whereas bryostatin, a differentiation blocker, only transiently activated ERK ( approximately 6 h) and attenuated the activation of ERK by TPA. To confirm a requirement for sustained ERK activation for megakaryocytic differentiation, PD098059, a synthetic inhibitor of the MAP kinase kinase 1 (MEK1) was employed. Introduction of PD098059 at any time during the first 18 h of TPA treatment completely abrogated megakaryocytic differentiation of K562 cells. After 24 h of TPA treatment, introduction of PD098059 failed to block differentiation. Differentiation blockade by PD098059 occurred via inhibition of MEK because transfection of a constitutively active mutant of MEK2 could override the PD098059 blockade. Experiments with conditioned media suggested that sustained activation of the ERK/MAP kinase pathway promoted the autocrine secretion of megakaryocytic lineage determination factors.

Sperm motility is regulated by protein phosphorylation. We have recently shown that a serine/threonine phosphatase system is involved in motility regulation. Two of the components of the phosphatase system, GSK-3 and PP1y2, are regulated by tyrosine phosphorylation. During our investigation of sperm tyrosine-phosphorylated proteins we discovered a 55-kDa protein whose tyrosine phosphorylation correlates closely to the motility state of sperm. This protein is tyrosine phosphorylated to a much higher degree in motile caudal than in immotile caput epididymal sperm. Motility inhibition of caudal epididymal sperm by protein kinase A (PKA) anchoring inhibition or by ionomycin-induced calcium overload led to the virtual disappearance of tyrosine phosphorylation of the 55-kDa protein. Conversely, treatment of sperm with motility activators, isobutylmethylxanthine or 8-bromo-cAMP, resulted in increased tyrosine phosphorylation of the protein. The protein was present in the soluble 100 000 x g supernatants of sperm extracts and was heat labile. Chromatography through diethylaminoethyl-cellulose and Western blot analysis showed that this 55-kDa protein is not a regulatory subunit of PKA or a-tubulin. Our results represent the identification of a soluble protein whose tyrosine phosphorylation varies directly with motility and suggest that motility regulation may involve cross talk between PKA, calcium, and tyrosine kinase pathways.

We have extensively purified three of the hepatic microsomal intralumenal Ca2+-binding proteins, CBP1, CBP2, and CBP3, which were originally described by Van et al. [(1989) J. Biol. Chem. 264, 17494-17501]. These apparently homogeneous preparations showed only single 45Ca2+ binding bands. On the basis of the peptide sequence, CBP2 was found to be highly homologous with the previously described protein ERp72. Similarly, CBP3 was identical to calreticulin and CBP1 had some homology to calmodulin. Contrary to the report of Van et al. (1989), we found that CBP2 had little thiol:protein disulfide oxidoreductase activity. Of the three purified preparations, only CBP2 exhibited apparent intrinsic protein kinase activity. This activity was found to be due to contamination of the CBP2 preparation by an extremely low concentration of tightly bound casein kinase 2 (CK2). In line with this observation, the phosphorylation was inhibited by heparin, removed by antibody to CK2, and stimulated by spermine. Furthermore, CBP2 was readily phosphorylated in vitro by added CK2 but only slowly phosphorylated by several other protein kinases. Thus, the persistence of CK2 in a highly purified preparation of CBP2 along with several other lines of evidence presented in this study might suggest that the protein CBP2 is a physiologically relevant substrate for CK2. Furthermore, these data suggest that CK2 might be localized in the lumen of the endoplasmic reticulum and that the phosphorylation of CBP2 in the lumen may play a role in the chaperone activity attributed to this protein.

The ecto-5’-nucleotidase (CD73) is glycosylphosphatidylinositol (GPI) anchored cell surface protein that is involved in switching on adenosinergic signaling. Its enzymatic and nonenzymatic activities (via its interaction with extracellular matrix components) are involved in cancer associated processes and not completely independent of each other. It catalyzes the hydrolysis of AMP into adenosine and phosphate, where adenosine plays important role in tumor immune escape. It is overexpressed in many types of cancer cell lines and patient's biopsies including breast, colon, ovarian, gastric, ovarian, etc. In addition to being important as clinical and prognostic marker in cancer patients, overexpression of CD73 is associated with resistance to antitumor agents, and inhibition of CD73 activity or knocking it down by SiRNA reversed chemoresistant phenotype of glioblastoma multiforme cells. Because of the significant role of CD73, it is of importance to develop an assay that monitor the activity of CD73 in order to develop modulators of its activity. Towards this goal, we have developed a bioluminescent assay to monitor the activity of the enzyme in biochemically pure as well cellular anchored enzyme forms. The assay is homogenous and formatted for HTS screening research, very sensitive, and robust as indicated by the high Z’. We also show that the use of inhibitors such as adenosine 5’-(α-β-methylene) diphosphate (APCP) generates data with biochemically pure enzyme similar to the cellular bound form. We also demonstrate that cells that are enriched in CD73 can be easily identified from those that have low CD73. Thus, the current assay is robust, sensitive, and easy to use making it an ideal assay for HTS screening for new modulators of CD73 and generation of potential novel cancer therapeutics Citation Format: Said A. Goueli, Kevin hsiao. Biochemical and cellular monitoring of the activity of the ecto-5’-nucleotidase (CD73), a key cancer modulator using HTS-formatted bioluminescent technology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 414.

JumonjiC domain-containing histone lysine demethylases (JMJCs) play a pivotal role in determining the epigenetic status of the genome, leading to either transcriptional repression or activation of target genes by counteracting the activities of histone lysine methyltransferases. Because of their implication in cancer, they have become validated drug targets. Therefore, assays for this enzyme subfamily are desirable in order to facilitate the identification of selective and potent inhibitors for drug discovery and as basic research tools. Since succinate is one of the products of the demethylation reaction catalyzed by these enzymes, an assay that detects succinate would be suitable for monitoring all JMJC activities as well as other succinate-forming enzymes (i.e.: dioxygenases). Thus, a bioluminescent and homogenous succinate detection assay for measuring JMJC activity was developed, and it is performed in a two-step format that relies on converting the succinate product to ATP, and then to light in a robust luciferase reaction. The light output is proportional to succinate concentration from low nM to 15μM, and the assay is highly sensitive and robust, two features that are highly desirable and essential for measuring the activity of the majority of JMJC demethylase subfamilies. Therefore, the succinate detection assay is a simple-to-use method that does not require antibodies or modified substrates. Examples of various applications of this succinate detection assay will be presented, including studies on specificity of different substrates by diverse JMJCs, as well as mode of action studies using specific inhibitors. The development of this succinate detection assay will make it possible to investigate a large number of JMJC demethylases and could have significant impact on diverse areas of Epigenetics research. Citation Format: Juliano Alves, Said Goueli, Hicham Zegzouti. A homogeneous bioluminescent succinate assay for detection of jumonjiC domain-containing histone demethylase activities. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 362.

Nucleolin is an abundant nucleolar phosphoprotein which has been implicated as a factor in various stages of ribosome synthesis, including transcription. Since androgens exert a profound effect on the rRNA synthesis in the target organ prostate, we have examined the nature of androgenic regulation of the amount and phosphorylation of nucleolin in this tissue. Phosphorylation of prostatic nucleolin is catalyzed in part by heparin-sensitive casein kinase 2 (CK-2) and by another (heparin-insensitive) protein kinase. Both the amount and phosphorylation of prostatic nucleolin are profoundly sensitive to androgens. Rapid reduction in the level and phosphorylation of nucleolin occurs following androgen deprivation, which corresponds to the ensuing cessation of prostatic growth leading to involution. Further, the loss of nucleolin phosphorylation and its degradation appear to be concordant. Administration of a single injection of 5α-dihydrotestosterone to castrated animals causes an early increase in the amount and phosphorylation of nucleolin, starting in the prereplicative phase in the prostatic cell nucleus. These data suggest that early androgenic regulation of nucleolin expression and phosphorylation may play a role in nucleolar control mechanisms relevant to prostatic cell growth. © 1994 Wiley-Liss, Inc.

In previous work, we had observed that chromatin-associated nonhistone protein phosphorylation, catalyzed by intrinsic protein kinase reaction in chromatin preparations from human benign prostatic hyperplasia (BPH) prostate samples was markedly elevated, compared with the normal prostate chromatin samples [Rayan et al: Cancer Res 45:2277-2282, 1985]. The properties of this protein kinase reaction were suggestive of the involvement of casein kinase(s). By employing the specific synthetic substrate for casein kinase 2 (CK-2) for assays in cellular fractions, we have shown that this protein kinase is present in human prostate chromatin. Its activity is increased in BPH chromatin by about 25-fold, as compared with its activity in the normal prostate chromatin. This suggests that CK-2 is a possible mediator of the enhanced phosphorylation of chromosomal proteins in BPH chromatin. By comparison, CK-2 activity in chromatin preparations from prostatic carcinoma samples was markedly less elevated than that of the BPH chromatin. Immunohistochemical analysis of the enzyme in human frozen sections of prostate tissue samples showed that the enzyme immunostaining was diffuse in the cytoplasm, but more intense in the nucleus, especially in the nucleoli. In general, the staining corresponded with the enzymic data. However, sections from prostatic carcinoma samples appeared to show differential staining, depending on the Gleason's grade of the sample. The samples with higher Gleason's grade showed less intense immunostain in the nucleus, compared with samples of lower Gleason's grade. Further, regions of sections in samples with higher Gleason's grade did not show any immunostaining. These differences in the characteristics of CK-2 expression in prostatic carcinoma samples may be potentially significant, but need to be evaluated further for their significance to the pathobiology of prostatic neoplasia.

Casein kinase 2 (CK-2) is a ubiquitous messenger-independent protein serine/threonine kinase that has been implicated in growth control. We have studied the activity and subcellular location of CK-2 in adult rat ventral prostate in relation to androgen withdrawal and administration. Androgen deprivation by castration results in a faster decline in CK-2 activity associated with prostatic nuclei than that in the cytosol. Nuclear CK-2 associated with chromatin is reduced at an even greater rate than that in the total nucleus. Reversal of these events by administration of a single dose of 5 alpha-dihydrotestosterone to adult rats castrated 144 hr previously was accompanied by a differential early enhancement of chromatin-associated CK-2 activity, with a concomitant decrease in the CK-2 activity present in the cytosol. Changes in the nuclear CK-2 activity correlated with the immunostainable enzyme protein in the nucleus. We propose that androgens evoke translocation of CK-2 from the cytoplasm to the nucleus (nucleoplasm) where its enhanced association with the chromatin constituents takes place. Conversely, withdrawal of circulating androgens due to castration evokes a dissociation of CK-2 from chromatin and eventual translocation of nucleoplasmic CK-2 to the cytoplasm. Modulations in the association of CK-2 with nuclear chromatin may represent an important mechanism of post-transcriptional regulation of nuclear CK-2 in relation to androgen action in the prostate.

A novel heat-stable cellular protein that significantly stimulated Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) was identified. It is ubiquitous and has a molecular mass of over 150 kDa, and was shown to be specific for PKC. It activates PKC in the absence and presence of phospholipids; however, its maximal stimulatory potential was achieved when optimal amounts of phospholipids were also present in the reaction medium. Thus, in comparison with classical cofactors such as phospholipid, where activation of PKC mainly involves interaction with the regulatory domain of PKC, the mechanism of activation of PKC by the activator appears to involve several binding sites.

Post transcriptional modifications of proteins and nucleic acids are well-recognized to play major role in mediation of extensive and broad range of cellular functions. Such modifications that include phosphorylation, acetylation, methylation, etc. are known to be critical for normal and abnormal cell growth, and thus, the enzymes involved in these reactions have been validated as drug targets resulting in the development of several drugs. Recent biochemical and biological data suggest that the enzymatic activities of several of these enzymes have pathogenic roles in cancer, inflammation, and neurodegenerative diseases. Thus, pharmacological modulation of these enzymes by small molecules will be beneficial in the development of novel therapeutics for multiple unmet medical needs. Of these, methyltransferases are known to alter the epigenome by altering the methylation status of nucleic acids or proteins resulting in changes in cellular phenotypes. 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. The assay is universal 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, protein, RNA, and small molecules methyltransferases. 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 and low C.V. The assay is 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 with no limitations on the use of high concentrations of substrates 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. Monitoring the activity of various classes of methylltransferases in vitro using a universal, homogenous and bioluminescent assay. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A31.

A monoclonal antibody was produced against the nuclear casein kinase II (PK-N2)* isolated from rat liver. The antibody was of the IgM class, and showed immunoreactivity towards the higher molecular weight subunit (41K Da) of the protein kinase in Western blots. The antibody was equally reactive towards the PK-N2 isolated from rat ventral prostate indicating that it can recognize the enzyme from different tissues of the rat. The antibody also detected the cytosolic casein kinase II (CK-2) suggesting significant similarity of the antigenic domains in the two forms of this protein kinase. No binding was detected with the nuclear or cytosolic casein kinase I (PK-N1 and CK-1). The antibody did not inhibit the enzyme activity or directly precipitate the enzyme, but when coupled to an affinity matrix and cross-linked with dimethylpimelimidate, it was capable of removing nearly all the PK-N2 activity from solution.

An age-associated decline in the activity of cytosolic casein kinase II in the rat ventral prostate was observed. The decrease in specific and total activity of casein kinase II of prostatic cytosol obtained from 12-month old rats compared with that from 3-month old rats was 50% and 70%, respectively. This decrease was tissue specific since no such alteration in enzymic activity was found in liver, lung, or heart. The decline in activity was not due to an increase in the concentration of a casein kinase inhibitor or to altered androgenic status with aging. Rather, the reduction in the activity of the enzyme was commensurate with the decrease in the total concentration of the enzyme protein as determined by an ELISA using anti-casein kinase II antibodies. The activity of casein kinase II in the nuclear fraction of the rat prostate was not altered in aging animals. It may be concluded that the cytosolic form of casein kinase II in the prostate of older animals is regulated at the transcriptional level (but not via the androgen-receptor complex mechanism). An alternative explanation for this observation may relate to altered stability of the enzyme in the prostate of older animals.

We have developed a novel assay for monitoring changes in intracellular cyclic AMP (cAMP) concentration with high sensitivity (30 +/- 5 fmol [mean +/- standard error of the mean] of cAMP per well) and reproducibility (Z' of > 0.8). The assay is of format amenable to high throughput screening (HTS) in 96-, 384-, and 1,536-well plates, and as a bioluminescent assay is potentially less prone to interferences originating from fluorescent compounds. Because of its high sensitivity, fewer numbers of cells (1,000 cells per well) in low-volume 384-well plates are required to screen for changes in cAMP concentrations. The assay does not rely on the use of antibodies, and thus it does not suffer from changes in the affinity or quality of the antibodies. The assay is based on the fact that cAMP is a potent activator of cAMP-dependent protein kinase (PKA), and activation of PKA can be monitored by measuring ATP utilization in a kinase reaction. The amount of ATP consumed can be measured using a luciferase/luciferin luminescent reaction. Since the amount of relative luminescence units (RLU) generated is a measure of the remaining ATP, a reciprocal relationship between RLU and both the activity of PKA and the intracellular concentration of cAMP is observed. Thus, the functional activity of agents that modulate the activity of Galpha(s) or Galpha(i) forms of G-protein-coupled receptors (GPCRs), which cause change in intracellular cAMP, can be monitored by the change in the activity of PKA and the amount of RLU readout. The assay can be performed in two steps and requires only 30 min after cell lysis for completion. The assay has been successfully used to generate 50% effective concentration (EC(50)) values for forskolin, a known direct activator of cellular adenylate cyclases, and EC(50) values for agonists and 50% inhibitory concentration values for antagonists modulating GPCRs that alter adenylate cyclase activity (Galpha(s) and Galpha(i)). Finally, adherent, suspension, and frozen cells have been successfully used in this assay, thus offering flexibility and convenience for many HTS applications.

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 transcriptional modifications of proteins and nucleic acids are well-recognized to play major role in influencing cell fate towards cellular proliferation, differentiation, and cell death. Such modifications that include phosphorylation, acetylation, methylation, etc. have been validated as drug targets resulting in the development of several drugs. Recent biochemical and biological data suggest that the enzymatic activities of several of these enzymes have pathogenic roles in cancer, inflammation, and neurodegenerative diseases. Thus, pharmacological modulation of these enzymes by small molecules will be beneficial in developing novel therapeutics for multiple unmet medical needs. Of these, methyltransferases are known to alter the epigenome by altering the methylation status of nucleic acids or proteins resulting in changes in cellular functions. 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. The assay is universal 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, protein, RNA, and small molecules methyltransferases. 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 and low C.V. The assay is 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 with no limitations on the use of high concentrations of substrates 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: Kevin Hsiao, Said Goueli. Universal homogenous bioluminescent assay that monitor the activity various classes of methyltransferases in vitro. [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 93. doi:10.1158/1538-7445.AM2015-93

GTPases play a major role in various cellular functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators have been challenging due to lack of convenient assays. To overcome the challenges in analyzing activities of GTPases and their regulatory proteins GTPase Activating Proteins (GAP) and Guanine Nucleotide Exchange Factors (GEF) we have developed a homogenous bioluminescent assay (GTPase/GAP/GEF-Glo) system to analyze these proteins in a simple, convenient “add-mix-read” format. Mg2+ plays a critical role in influencing affinity of GTPases with GTP/GDP and the process of nucleotide exchange. Manipulating Mg2+ concentrations in the GTPase reaction buffer allows continuous progression of the GTPase cycle and faster hydrolysis of GTP. The assay relies on enzymatic conversion of GTP remaining after the GTPase reaction to ATP and detection of the ATP using luciferin/luciferase combination. We show that the assay is sensitive, and robust when analyzing for analyzing intrinsic GTPase- activity, GAP-stimulated GTPase- activity, GAP-activity and GEF- activity. The system can also be used to analyze these proteins expressed in cells as a fusion protein performing the assay in a pulldown -format. Furthermore, there was minimal false hits when tested for compound interference using the library of pharmacologically active compounds (LOPAC) and its robustness as indicated by high Z’-factor of 0.93 and CV of 2.2%. Citation Format: Subhanjan Mondal, Kevin Hsiao, Said A. Goueli. A homogenous bioluminescent system for measuring GTPase, GAP and GEF activities. [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 3924. doi:10.1158/1538-7445.AM2015-3924

To establish a cell culture system with long-term replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro.HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naive cells. The presence of minus (antisense) RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques (flow cytometry and Western blot) respectively.The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-E1). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells.HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.

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.

Protein kinases and phosphatases play an important role in a variety of cellular functions such as cell growth, development, and gene expression (1). It is estimated that one-third of the proteins in a typical mammalian cell are phosphorylated and about 200 protein kinases and 100 protein phosphatases have been identified. In addition, perhaps 2-3% of the genes in the entire genome of an eukaryotic cell may code for protein kinases and as many as 5% of the human genes may encode protein kinases and phosphatases (2). The fact that these protein kinases and phosphatases have multiple substrates in vivo may explain their diverse physiological functions (1-3). Thus, it is of considerable interest to develop an assay system that is specific for certain protein kinases and simple enough to be used by both the novice as well as the expert in the field.

Protein kinases play an important role in many cellular processes and mediate cellular responses to a variety of extracellular stimuli. They have been identified by many pharmaceuticals as valid targets for drug discovery. Because of the large number of protein kinases, and the large number of compounds to be screened, it is important to develop assay systems that are not only sensitive but also homogeneous, fast, simple, nonradioactive, and cost-effective. Here we present a novel, rapid, robust assay to measure the enzyme activity of low concentrations of several serine/threonine and tyrosine protein kinases. It is based on the use of fluorogenic peptide substrates (Rhodamine 110, bis peptide amide) that are cleaved before phosphorylation to release the free Rhodamine 110; upon phosphorylation, cleavage is hindered, and the compound remains as a nonfluorescent peptide conjugate. The assay can be carried out in single- as well as multiwell plate formats such as 96- and 384-well plates. The signal-to-noise ratio is very high (40), the Z ′ is over 0.8, and the signal is stable for at least 4 h. Finally, the assay is easily adapted to a robotic system for drug discovery programs targeting protein kinases.

Introduction: Hepatitis C virus (HCV) is a single strand RNA hepatotrophic virus infecting 170 millions around the world and 20% of Egyptian blood donors. Although there has been significant improvement in the enzyme immunoassays (EIAs) in population screening of HCV infection, the development of a low variability, easy to automate and inexpensive supplemental test to support the current immunoassays was of a major concern to several laboratories. Objectives: In the current study, we embarked on a systematic study to analyze by DNA sequencing several HCV isolates to identify conserved core protein sequences and perform explorative analysis of five synthetic peptides from the core/E1 region in anti-HCV antibody assays. Methods: We designed four synthetic-core specific peptides and an E1-specific peptide. These peptides were used to screen HCV antibodies in sera of 100 HCV positive patients and 100 HCV negative subjects and compared the results with those obtained by the commercial systems based on second and third generation enzyme-linked immunosorbent assays. Results: Our results showed that all peptides detect HCV antibodies in infected sera to varying degrees. The synthetic peptide (a.a. 21–40) of the core protein had 99% sensitivity, 100% specificity and was highly reproducible. Conclusion: The above findings make this core peptide a candidate product for developing a supplemental test for chronic HCV infection in the Egyptian population.

It is well known that GTPases of the Ras superfamily play major roles in cell functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators have been challenging due to lack of convenient assays. We have developed a homogenous bioluminescent assay for monitoring the activities of GTPase and its immediate regulators: GAP and GEF. Mg2+ plays a critical role in influencing affinity of GTPases with GTP/GDP. By manipulating Mg2+ concentrations in the reaction buffer allows continuous progression of the GTPase cycle and hydrolysis of GTP. The assay relies on the conversion of remaining GTP to ATP and detection of the latter using luciferin/luciferase combination. The GTPase/GAP/GEF-Glo™ assay system enables monitoring of GTPase-, GAP-stimulated GTPase-, GAP- and GEF- activities. The assay system has a high dynamic range and signal-to-background ratio; it is in a convenient “add-mix-read” format and applicable to high throughput screening. Citation Format: Subhanjan Mondal, Said A. Goueli. GTPase/GAP/GEF-Glo™: A bioluminescent system to measure GTPase, GAP, and GEF activities. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B44. doi: 10.1158/1557-3125.RASONC14-B44

Adenine nucleotides are major determinants of the energy status of the cell and thus any modulation of their cellular concentration has significant consequences to cellular metabolism, cellular growth and cell death. Many biochemical reactions that generate AMP as a reaction product are pivotal players in a variety of signaling pathways and thus are considered validated drug targets. These include enzymes involved in protein ubiquitination (Ubiquitin ligases), protein synthesis (aminoacyl tRNA synthetases), second messenger signaling (cAMP-phosphodiesterases), and DNA synthesis and repair (prokaryotic and eukaryotic DNA ligases). Therefore, an assay that monitors the activity of these enzymes is desirable in the search for selective modulators and the development of novel therapeutics. As AMP is a common product of these enzymatic reactions, the development of an assay that monitors AMP in a homogenous and sensitive fashion will have significant impact in these diverse areas of research. In addition, enzyme reactions that generate products which can be converted to AMP can be also monitored using this assay. We demonstrate the utility of this assay platform in monitoring the activity of cAMP-PDE, aminoacyl tRNA synthetases, ubiquitin ligases, DNA ligases. Furthermore, enzyme reactions such as methyltransferases that produce product (SAH) which can be converted to AMP can be monitored using this assay platform Citation Format: Said A. Goueli, Kevin Hsiao, Hicham Zegzouti, Subhanjan Mondal. Monitoring the level of the universal nucleotide AMP in diverse enzymatic reactions using bioluminescent homogenous assay platform. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3735. doi:10.1158/1538-7445.AM2014-3735

Publisher Summary The phosphorylation of cellular proteins or enzymes may result in alteration of their conformation and/or their enzyme activity. This is best illustrated by a group of enzymes collectively known as mitogen-activated protein (MAP) kinases, whose dual phosphorylation by upstream protein kinases results in their activation. These kinase cascades are found in all eukaryotic organisms and consist of a three-kinase module. The three modules contain the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK (p38) pathways. This chapter describes the phospho-specific mitogen-activated protein kinase antibodies for extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 activation. The availability of antibodies that recognize the active form of the various members of MAPKs (ERKs, JNKs, and p38) has made it possible to study the activation of these enzymes in response to a variety of stimuli in an accurate and time-dependent fashion. Similar to the anti-phosphotyrosine antibodies, the dual phospho-specific antibodies have proved to be valuable reagents in the pursuit of selective inhibitors of these enzymes. Studies of these enzymes with these novel antibodies also resulted in the discovery of novel MAPK kinase kinase (MEK) inhibitors such as U0126.

Publisher Summary This chapter discusses assaying activity of individual protein kinases in crude tissue or cellular extracts. Protein kinases and phosphatases play an important role in a variety of cellular functions such as cell growth, development, and gene expression. It is estimated that about 2-3% of the genes in the entire genome of a eukaryotic cell may encode protein kinases and as many as 5% of human genes may encode protein kinases and phosphatases. The fact that these protein kinases and phosphatases have multiple substrates in vivo may explain their diverse physiological functions. The availability of peptides that serve as specific substrates for certain protein kinases made it possible to determine the activity of a specific protein kinase in a tissue or cellular extract with minimal interference from other enzymes. A widely used method to monitor the phosphopeptide product is the negatively charged phosphocellulose P-81 method, which requires the substrate to contain at least two or three basic amino acids, because the binding is based on electrostatic interaction.

Multiple isozymes of Ca2+/phospholipid-dependent protein kinase (PKC) were isolated from the rat ventral prostate. The enzyme exists mainly as type II (beta), and type III (alpha) forms, and it is possible that type II isozyme may comprise the subspecies beta 1 and beta 2. The total and specific activities of prostatic PKC isoforms were reduced in castrated animals; this decrease was specific since administration of androgens to castrated animals reversed such a decline. Also, there was a differential response to androgen deprivation so that type III isozyme declined at a faster rate than that of type II. Thus, our studies show for the first time that PKC of the rat ventral prostate comprises multiple isozymes, and that the activity of these various forms are differentially regulated by androgens.

Integrins play a critical role in cell adhesion and mediate cell signaling. This report identifies the association of serine protein kinase activity with the beta 1 integrin by immunoprecipitation and phosphoamino acid analysis techniques. Reprecipitation techniques suggested that the serine kinase activity was not a member of the protein kinase C family. By gel filtration, most of the protein kinase activity associated with beta 1 integrin as well as most of the cell-surface beta 1 integrin was present in large detergent resistant complexes. These results suggest that serine protein kinase activity associated with the beta 1 integrin may play a role in signaling via the beta 1 integrin.

Cyclic AMP-dependent protein kinase (PKA) is anchored at specific subcellular sites through the interaction of the regulatory subunit (R) with protein kinase A-anchoring proteins (AKAPs) via an amphipathic helix binding motif. Synthetic peptides containing this amphipathic helix domain competitively disrupt PKA binding to AKAPs and cause a loss of PKA modulation of cellular responses. In this report we use S-Ht31, a cell-permeant anchoring inhibitor peptide, to study the role of PKA anchoring in sperm. Our analysis of three species of mammalian sperm detected three isoforms of PKA (RIIalpha, RIIbeta, and RIbeta) and one 110-kDa AKAP. The addition of S-Ht31 to bovine caudal epididymal sperm inhibits motility in a time- and concentration-dependent manner. A control peptide, S-Ht31-P, identical to S-Ht31 except for a proline for isoleucine substitution to prevent amphipathic helix formation, had no effect on motility. The inhibition of motility by S-Ht31 is reversible but only if calcium is present in the suspension buffer, suggesting a role for PKA anchoring in regulating cellular calcium homeostasis. Surprisingly, inhibition of PKA catalytic activity had little effect on basal motility or motility stimulated by agents previously thought to work via PKA activation. These data suggest that the interaction of the regulatory subunit of PKA with sperm AKAPs, independent of PKA catalytic activity, is a key regulator of sperm motility and that disruption of this interaction using cell-permeable anchoring inhibitor peptides may form the basis of a sperm-targeted contraceptive.

Methylation/demethylation of DNA and proteins play major roles in modulation of the epigenome and has been implicated in a wide variety of human diseases. Recent biochemical and biological data suggest that the enzymatic activities of several of these enzymes have pathogenic roles in cancer, inflammation, and neurodegenerative diseases. 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. The assay is universal 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, protein, and small molecules methyltransferases. 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 and low C.V. The assay is 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 with no limitations on the use of high concentrations of substrates 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 Information: Mol Cancer Ther 2013;12(11 Suppl):A125. Citation Format: Said A. Goueli, Kevin Hsaio. Universal homogenous bioluminescent assay to monitor the effect of modulators on various classes of methyltransferases in vitro. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A125.

It is well known that GTPases play major roles in cell functions such as GPCR signaling, cell proliferation, cell differentiation, cytoskeleton modulation, and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators have been challenging due to lack of convenient assays. GTPases act as molecular switches cycling between an activated GTP-bound state (“ON”-state) and an inactive GDP-bound state (“OFF”-state). They have very high affinity for guanine nucleotides GDP or GTP and are extremely slow acting GTP-hydrolases. To mediate the process of shuttling between the active and inactive forms, GTPases are modulated by two groups of enzymes, guanine nucleotide exchange factors (GEF) and guanine nucleotide activating proteins (GAPs). The GEFs proteins substitute GTP for the bound GDP and thus activating them while the GAP family activates the hydrolysis of GTP and terminating their effector function. We have developed a homogenous bioluminescent assay for monitoring the enzyme activity of GTPase, GAP, and GEF proteins. Mg2+ plays a critical role in influencing affinity of GTPases with GTP/GDP, and manipulating Mg2+ concentrations in the reaction buffer allows continuous progression of the GTPase cycle and hydrolysis of GTP. The assay relies on the conversion of remaining GTP to ATP and detection of the latter using luciferin/luciferase combination. The assay system for measuring GTPase/GAP/GEF-Glo™ is enabling to monitoring the intrinsic GTPase-, GAP-stimulated GTPase-, GAP-, and GEF- activities. The assay system has a high dynamic range and signal-to-background ratio; it is in a convenient “add-mix-read” format and applicable to high throughput screening. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C204. Citation Format: Subhanjan Mondal, Said Goueli, Kevin Hsiao. GTPase/GAP/GEF-Glo™: A bioluminescent system to measure GTPase, GAP, and GEF activities. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C204.

Methylation/demethylation of DNA and Proteins play major roles in modulation of the epigenome and has been implicated in a wide variety of human diseases. Recent biochemical and biological data suggest that the enzymatic activities of several of these enzymes have pathogenic roles in cancer, inflammation, and neurodegenerative diseases. 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. The assay is universal 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, protein, and small molecules methyltransferases. 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 and low C.V. The assay is 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 with no limitations on the use of high concentrations of substrates 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, Hicham Zegzouti. Universal homogenous bioluminescent assay to monitor the effect of modulators on various classes of methyltransferases in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4236. doi:10.1158/1538-7445.AM2013-4236

Adenine nucleotides are major determinants of the energy status of the cell and thus any modulation of their cellular concentration has significant consequences to cellular metabolism, cellular growth and cell death. ATP generating enzymes are usually involved in anabolic processes while ATP consuming enzymes are involved in catabolite processes. We have developed biochemical assays that monitor the concentrations of ATP, ADP and AMP in a biochemical reaction. For instance ATP depletion dependent assays and ADP generating assays monitor the activity of kinases and ATPases. These universal assays can measure the activity of various enzymes with no modification of the native substrate and the ability to use diverse substrates such as proteins, peptides, lipids, sugars, etc. The AMP generating reactions such as ubiquitin ligases, aa aminoacyl t-RNA synthetases, DNA ligases, cAMP-dependent phosphodiesterases, etc. can be also monitored with high sensitivity and reproducibility. We will show data using these various technologies in monitoring the various adenine nucleotide concentrations in biochemical reactions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 164. doi:1538-7445.AM2012-164

Protein Kinases are implicated in metabolic and cellular disorders including cancer and thus they represent a very important target for the development of novel therapeutics. The process of developing new drug requires an innovative and low cost strategy to screen and identify compounds that can be used for lead optimization that incorporate the required desirable features. It will be idea to have a universal technology that can be used for the various stages of drug discovery such as assay development, compound screening, lead optimization, and enzyme profiling using the same technology platform. The technology should provide high sensitivity, robustness, and low cost. Towards that goal, we have expanded our luminescent platform for monitoring kinase activity to enable us to detect enzyme activity using low amount of enzymes, measure initial rates at very early substrate conversion, and generate reliable and robust data. This luminescent assay monitors enzyme activity by measuring ADP production using a homogenous and high through put formatted assay. The assay is also ideal for profiling compounds against various kinases since it detects ADP, the universal product of all kinases. Thus, the new homogenous assay is most suitable for drug discovery targeting kinases since it can be used in all various stages of drug development leading to animal or cellular studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5445. doi:10.1158/1538-7445.AM2011-5445

Because of its versatility (all types of substrates), robustness (Z’>0.8), rapid performance, and its ease of use, the luminescence based Kinase GloTM assays platform have gained wide acceptance in many drug screening programs for protein kinase inhibitors. We have attempted to extend our strategy by now monitoring ADP product formation in the kinase reaction. The new assay we developed (ADP Glo) maintains all the positive features of Kinase GloTM such as universality, and applicability to all kinds of kinase substrates regardless of their nature with no prior modification (peptides, protein, polymer, lipids, and sugars) but In stead of measuring ATP depletion, it monitors ADP production. It detects ADP at early stages of enzyme reactions with very high signal to background (SB) ratio. It can be run at a wide range of ATP concentrations (low micromolar to millimolar), is sensitive to low ADP concentrations (0.1 pmol), and it can be carried out in high density plate formats. This assay is robust and homogenous, and does not require antibodies or custom synthesized substrates. It is ideal to search for optimal substrates in a collection of peptides, proteins, or lipids in a single assay format, and to screen for ATP competitive and Non competitive inhibitors. Because of the high dynamic range and more importantly, the high SB values even at low % ATP to ADP conversion, the assay allows the use of low amounts of enzyme during high-throughput screening. Evaluation of this assay in a screen of a large chemical library showed minimal false hits attesting to the quality of this system. The ADP-GloTM assay is applicable to all types of kinases, ATPases, multidrug resistant proteins such as MDR1-Pgp, helicases, topoisomerases, and heat shock proteins and as well as many other ADP producing enzyme reactions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1048.

To investigate the effects of mutations in domain III of the hepatitis C virus (HCV) internal ribosome entry sequences (IRES) on the response of chronic HCV genotype 4a patients to interferon therapy.HCV RNA was extracted from 19 chronic HCV 4a patients receiving interferon/ribavirin therapy who showed dramatic differences in their response to combination therapy after initial viral clearance. IRES domain III was cloned and 15 clones for each patient were sequenced. The obtained sequences were aligned with genotype 4a prototype using the ClustalW program and mutations scored. Prediction of stem-loop secondary structure and thermodynamic stability of the major quasispecies in each patient was performed using the MFOLD 3.2 program with Turner energies and selected constraints on base pairing.Analysis of RNA secondary structure revealed that insertions in domain III altered Watson-Crick base pairing of stems and reduced molecular stability of RNA, which may ultimately reduce binding affinity to ribosomal proteins. Insertion mutations in domain III were statistically more prevalent in sustained viral response patients (SVR, n = 14) as compared to breakthrough (BT, n = 5) patients.The influence of mutations within domain III on the response of HCV patients to combination therapy depends primarily on the position, but not the frequency, of these mutations within IRES domain III.

Nuclei isolated from rat ventral prostate contain a number of messenger-dependent and -independent protein kinases. Studies were undertaken to determine the relative contribution of these protein kinases in phosphorylation of non-histone proteins (NHPs) in isolated nuclei. The data suggest that messenger-dependent protein kinases such as those dependent on cAMP or Ca2+/calmodulin or Ca2+/phospholipid may be present in very small amounts in intact isolated nuclei, and thus appear not to be significantly involved in phosphorylation of endogenous NHPs. Messenger-independent nuclear associated protein kinases PK-N1 and PK-N2 are known to catalyze the phosphorylation of NHPs in vitro (Goueli Sa, et al., Eur J Biochem 113: 45-51, 1980). Of these, the intrinsic heparin-sensitive PK-N2 as compared with heparin-insensitive PK-N1 appeared to be the predominant protein kinase engaged in phosphorylation of NHPs in intact nuclei. About 78-88% of NHP phosphorylation in intact nuclei was inhibited by heparin suggesting that the remaining 12-22% phosphorylation of NHPs was catalyzed via the heparin-insensitive protein kinase(s). Further, the data provide additional evidence that heparin-sensitive PK-N2 is the one that is most responsive to androgenic status in the animal.

Basic fibroblast growth factor (FGF) has recently been shown to be a phosphoprotein and its receptor affinity is modified by phosphorylation. Since most studies of protein phosphorylation have focused on intracellular protein kinases, a physiologic mechanism of the regulation of basic FGF activity by phosphorylation has been unclear. Evidence for the existence of ecto-protein kinase activity on the surface of several types of cells including human neutrophils has been described. These ecto-protein kinase activities utilize exogenous ATP to phosphorylate exogenous as well as endogenous cell-surface proteins. This report demonstrates that human basic FGF is phosphorylated on serine by human neutrophil ecto-protein kinase activity and this phosphorylation is inhibited by heparin. Regulation of basic FGF activity by phosphorylation may be one of the functions of ecto-protein kinase activity.

Background Hepatitis C (HCV) viral infection is a serious medical problem in Egypt and it has a devastating impact on the Egyptian economy. It is estimated that over 15% of Egyptians are infected by the virus and thus finding a cure for this disease is of utmost importance. Current therapies for hepatitis C virus (HCV) genotype 4 with interferon/ribavirin have not been successful and thus the development of alternative therapy for this genotype is disparately needed. Results Although previous studies utilizing viral subgenomic or full cDNA fragments linked to reporter genes transfected into adhered cells or in a cell free system showed promise, demonstration of efficient viral replication was lacking. Thus, we utilized HepG2 cells infected with native HCV RNA genomes in a replication competent system and used antisense phosphorothioate Oligonucleotides (S-ODN) against stem loop IIId and the AUG translation start site of the viral polyprotein precursor to monitor viral replication. We were able to show complete arrest of intracellular replication of HCV-4 at 1 uM S-ODN, thus providing a proof of concept for the potential antiviral activity of S-ODN on native genomic replication of HCV genotype 4. Conclusion We have successfully demonstrated that by using two S-ODNs [(S-ODN1 (nt 326–348) and S-ODN-2 (nt 264–282)], we were able to completely inhibit viral replication in culture, thus confirming earlier reports on subgenomic constructs and suggesting a potential therapeutic value in HCV type 4.