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APPROVED PMLSC ASSAY PROTOCOLS
  • PMLSC Assay Protocol Review Document for MKP-1 HTS
  • PMLSC Assay Protocol Review Document for Cdc25B Catalytic Domain HTS
  • GNHR Translocation Assay APAC Review document
  • Necroptosis PMLSC APAC Document
  • PLK-1 PBD PMLSC APAC Document
  • West Nile Virus NS2bNS3 PMLSC APAC Document
  • Time Resolved Fluorescence Resonance Energy Transfer assay for Plk1 inhibitors
  • PKD IMAP TR-FRET APAC Document
  • High-content cell-based screening for modulators of autophagy
  • MKP-3 Chemical Complementation assay
  • PMLSC PROBE REPORTS
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    Approved PMLSC Assay Protocols
     
    bullet point  PMLSC Assay Protocol Review Document for MKP-1 HTS
     
     
    The mitogen-activated protein kinases (MAPK) are members of the signaling cascades for diverse extracellular stimuli that regulate fundamental cellular processes including embryogenesis, differentiation, mitosis, apoptosis, movement and gene expression. Four distinct MAP kinase families have been described; the extracellular signal-regulated kinases (ERK’s), c-jun N-terminal (JNK) or stress-activated protein kinases (SAPK), ERK5/big MAP kinase 1 (BMK1), and the p38 group of protein kinases. Since MAPK’s are regulated by phosphorylation, it therefore follows that phosphatases will be a key element of their control, and a family of dual specificity (Ser/Thr/Tyr) MAPK phosphatases (MKP1, MKP2 and MKP3) have been implicated. Mitogen-activated protein kinase phosphatase-1 (MKP-1, DUSP1 or CL100) dephosphorylates and inactivates MAPK substrates, such as p38, JNK, and Erk, and has been implicated in neoplasia. The MKP-1 assay is a homogeneous fluorescence intensity assay measuring the hydrolysis of the generic substrate 3-O-methylfluorescein phosphate (OMFP). We report here the optimization that was required for the development of a 384-well HTS assay that conforms to the PMLSC assay development and implementation guidelines.
     
    Documents
  • PMLSC Assay Protocol Review Document for MKP-1 HTS
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    bullet point  PMLSC Assay Protocol Review Document for Cdc25B Catalytic Domain HTS
     
     
    Cdc25 is a dual specificity protein tyrosine phosphatase that plays a pivotal role in the regulation of the cell cycle. Of the three isoforms that exist (Cdc25A, B, and C), Cdc25A and Cdc25B have been found to be oncongenic and over-expressed in many cancer cell lines. Cdc25B has been a target in multiple drug discovery endeavors, leading to several inhibitors of phosphatase activity in the micromolar to sub-micromolar range. Many of these compounds have been shown to induce G2/M cell cycle arrest with anti-proliferative effects. The Cdc25B catalytic domain assay is a homogeneous fluorescence intensity assay measuring the hydrolysis of the generic substrate 3-O-methylfluorescein phosphate (OMFP). We describe here the optimization that was required for the development of a 384-well HTS assay that conforms to the PMLSC assay development and implementation guidelines.
     
    Documents
  • PMLSC Assay Protocol Review Document for Cdc25B Catalytic Domain HTS
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    bullet point  GNHR Translocation Assay APAC Review document
     
     
    Cytoplasmic dynein is the molecular motor that carries cargo to the minus ends of microtubules (MTs) (e.g., from the cytoplasm to the nucleus), and provides the mechancial force for many other important fuctions, including nuclear envelope breakdown and sister chromatid exchange at mitosis. Unlike the numerous MT plus end-directed molecular motors, the kinesins, no specific small molecule inhibitors of dynein are known. Weak and nonspecific redox perturbers and nonspecific mimics of ATP (e.g., e/ytf""o-9-hydroxynonyladenine, aka EHNA) or phosphate anion
    (e.g., vanadate) are the only known dynein inhibitors. New inhibitors with potency and specificity would be invaluable cell biology tools. This void in our chemical biology toolbox is due in large part to both the large size of the work-performing component of the complex, dynein heavy chain (DYNC1H1) and the working complex itself, as well as the relative difficulty of working with functional dynein from biological systems. The overall goal of this work is to develop a refined suite of high throughput cell and biochemical assays for screening of chemical libraries to find experimentally useful inhibitors of cellular cytoplasmic dynein.
     
    Documents
  • PMLSC APAC Reports\GNHR Translocation Assay APAC Review document
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    bullet point  Necroptosis PMLSC APAC Document
     
     
    Necrosis is a caspase-independent cell death marked by a rapid loss of plasma membrane integrity, organelle swelling and mitochondrial dysfunction, and lacking typical features of apoptosis such as internucleosomal DNA cleavage and nuclear condensation. Necrosis has been reported to occur under a variety of pathological conditions, including both acute and chronic neurodegenerative disorders (8, 9) and may also occur during normal development of the nervous system (10, 11). Recent studies have found the classical morphological features of necrosis in the developing nervous system of apaf-1-/-, caspase-3-/- and caspase-9-/- mice which lack a part of the core apoptotic machinery (12). While necrosis has traditionally been viewed as an uncontrolled pathologic cell death, these studies support the concept that cells possess a caspase-independent but regulated cell death mechanism distinct from that of apoptosis, and the activation of this alternative mechanism leads to cell death with features resembling necrosis.
     
    Documents
  • Necroptosis PMLSC APAC Document
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    bullet point  PLK-1 PBD PMLSC APAC Document
     
     
    The Polo-like kinases (Plks) play important roles in many cell cycle-related events including the initiation of mitosis, chromosome segregation, centrosome maturation, bipolar spindle formation, regulation of anaphase-promoting complex and execution of cytokinesis. The prototypic Polo kinase was originally identified in flies, in which mutants resulted in abnormal spindle poles. A single Polo family member is found in flies, budding yeast, and fission yeast (Polo, CDC5 and Plo1, respectively), while 3 Plk family members are found in humans, mice and frogs (Plk1/Plx1, Plk2/Snk/Plx2 and Plk3/Fnk/Prk/Plx3 respectively). One human family member, Plk1, as well as its homologues in Xenopus (Plx1) and budding yeast (Cdc5) plays a particularly important role in coordinating multiple events during mitosis. Plk1/Plx1/Cdc5 is critical for proper centrosome maturation and spindle assembly. Plk1/Plx1/Cdc5 protein expression peaks during late G2 and M phases, where it localizes to centrosomes and spindle pole bodies during prophase and metaphase. During this time, Plk1/Plx1 likely phosphorylates a number of key centrosome-associated proteins, including the mitotic phosphatase Cdc25C to initiate the Cdc25C-Cdc2 amplification loop at the onset of M phase. At metaphase, Plk1 activates the anaphase-promoting complex (APC) to upregulate the ubiquitin-dependent proteolytic degradation of factors that regulate passage through mitosis. In addition, Plk1 and its homologues play important roles in the later stages of mitosis, particularly in anaphase and cytokinesis. In budding yeast, for example, Cdc5 phosphorylates the Scc1 subunit of the cohesin complex, which holds sister chromatids together until the start of anaphase. Phosphorylation of Scc1 by Cdc5 facilitates proteolytic cleavage of Scc1 by separase to allow sister chromatid separation upon anaphase onset. Plk1 re-localizes to the spindle midzone during late anaphase, possibly playing roles in regulating components of microtubules and kinetochores, and subsequently re-localizes to the mid-body during telophase and cytokinesis, where it appears to play a critical role in formation of the contractile ring at the site of cytoplasmic division.
     
    Documents
  • PLK-1 PBD PMLSC APAC Document
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    bullet point  West Nile Virus NS2bNS3 PMLSC APAC Document
     
     
    West Nile virus, a member of the Flaviviridae family, was first isolated in 1937 in the West Nile district of Uganda. West Nile virus is transmitted to animals including humans, through mosquito bites. Mosquitoes become infected when they feed on infected birds. In 2003, West Nile virus was detected in as many as 46 of the United States. The virus infected as many as 10,000 people and was the cause of approximately 300 deaths. The data for 2004 are even more troubling because the virus has spread and intensified throughout the US. West Nile virus is also a potential bioterrorism weapon. West Nile virus, Dengue and Yellow Fever flaviviruses are Priority Pathogens according to the classification issued by the NIAID.

     
    Documents
  • West Nile Virus NS2bNS3 PMLSC APAC Document
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    bullet point  Time Resolved Fluorescence Resonance Energy Transfer assay for Plk1 inhibitors
     
     
    Polo-like kinase 1 (Plk1) is a serine-threonine protein kinase that functions as a key regulator of mitosis/meiosis and cytokinesis (Barr et al., 2004). Numerous research studies have demonstrated that Plk1 gene expression is frequently up-regulated in human cancers and carcinoma-derived cell lines (Simizu and Osada, 2000). Plk1 expression levels are abnormal in many spontaneous human cancers including pancreatic, ovarian, breast, non-small cell lung carcinoma, prostate, melanoma, colorectal, and gliomas (Weichert et al., 2004; Weichert et al., 2005; Takai et al., 2005). Interest in Plk1 is based on evidence that Plk1 inhibition by Plk1-specific anti-sense oligonucleotides, siRNA, or short hairpin RNA results in decreased tumor-derived cell survival and inhibited tumor growth in animal models (Spankuch-Schmitt et al., 2002; Spankuch et al., 2004; Guan et al., 2005). These data suggest that Plk1 is a rational and validated anticancer drug target. Interestingly, there is evidence that Plk1 is a molecular target for other diseases and/or viral infections. For example, it has been shown that Plk1 protein is (1) along with other cell cycle proteins, detectable in Alzheimer’s, but not in control neurons (Harris et al., 2000; Husseman et al., 2001); (2) up-regulated in CD4+ T cells from rhesus macaques infected with high viral load SIV (Bostik et al., 2004); (3) up-regulated in primary human kertinocytes infected with human papilloma virus type 16 E6 and E7 (Patel et al., 2004); and (4) is specifically associated with and/or phosphorylates the human cytomegalovirus pp65 lower matrix protein upon infection of human fibroblasts and HeLa cells (Gallina et al., 1999). While the significance of these latter findings is undefined, it suggests that aberrant Plk1 expression may result in unregulated cell cycle events leading to cell degeneration or that pathogenic viruses express and/or recruit Plk1 for their own benefit.
     
    Documents
  • Time Resolved Fluorescence Resonance Energy Transfer assay for Plk1 inhibitors APAC Document
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    bullet point  PKD IMAP TR-FRET APAC Document
     
     
    Diacylglycerol (DAG) is (1) a key second messenger in cells and (2) generated upon G-protein-coupled and tyrosine kinase receptor engagement/activation. There are currently six classes/families of DAG receptors: (1) Protein kinase C (PKC); (2) the chimerin Rac GTPase-activating proteins; (3) the Ras guanyl-nucleotide-releasing proteins (Ras GRPs); (4) Munc13s; (5) the DAG kinases (DGKs); and (6) Protein kinase D (PKD).
    There are four members within the PKD family: PKD1 (murine PKD, human PKCμ), PKD2 and PKD3 (PKDν). Although originally classified as an atypical isoform of the PKC, PKD/PKCμ actually belongs to a subfamily of serine/threonine kinases within the Ca2+/calmodulin-dependent kinase (CaMK) superfamily (see Wang 2006 for review). This is based primarily on the fact that PKD/PKCμ shares the highest sequence homology within its catalytic domain with myosin light chain kinase and CAMKs (Valverde et al., 1994). Interestingly, PKD/PKCμ serves as both a DAG receptor and a substrate for PKC (reviewed in Wang 2006). In an unactivated state, PKD/PKCμ is primarily localized in the cytosol; however, activated PKD/PKCμ is mobile and can move between different subcellular compartments. This ability of PKD/PKCμ to localize to different subcellular compartments suggests that the (1) localization of PKD/PKCμ at different sites results in different physiological functional roles; (2) shuttling of PKD/PKCμ between multiple subcellular compartments facilitates the transmission of signals between those compartments; and (3) migration of PKD/PKCμ between multiple subcellular compartments can localize PKD/PKCμ to different substrates, reminiscent of the localization of Polo-like kinase I to multiple mitotic structures during the cell cycle (reviewed in Wang, 2006; Marklund et al., 2003; Baron and Malhotra, 2002).
    There is considerable evidence emerging that PKD/PKCμ regulates a variety of physiological processes including signal transduction, membrane trafficking, cell survival, cell migration, cell differentiation and cell proliferation (Rozengurt et al., 2005; Wang 2006). As described in Wang et al. (2006), there is accumulating evidence that PKD/PKCμ may be a focus of future drug design efforts and the rationale is as follows:
    • PKD/PKCμ lies at a significant juncture of DAG-PKC signaling pathways which is often unregulated in cancer and other disease states
    • PKD/PKCμ is involved in DNA damage, cell growth, differentiation, survival, migration and invasion
    • PKD/PKCμ regulates the function of class IIa HDACs, which are drug targets of several disease states including cardiomyopathy, osteodystrophy, neurodegenerative disorders and cancer
    • PKD/PKCμ is involved in oxidative stress response, which may be involved with malignant transformation and aging
     
    Documents
  • PKD IMAP FP APAC Document
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    bullet point  High-content cell-based screening for modulators of autophagy
     
     
    The High-content cell-based assay for screening modulators of autophagy is the resultof a joint development project between Xiao-Ming Yin at the Department ofPharmacology and Andreas Vogt, UPDDI. The assay was jointly developed andoptimized and submitted to the NIH in response to PAR -06-545 (PI : Yin). Theapplication (1R03 MH-083154-01) received a priority score of 159 (16.9th percentile) on
    August 10, 2007 and was awarded to Dr. Yin on September 26, 2007. The applicationrequested to be assigned to the PMLSC. The NIH has extended a request to file an MTA to Dr. Yin the first week of September, 2007.
    We report here the assay optimization that was required to evaluate the suitability for HTS of the High-content cell-based screening for modulators of autophagy according to
    UPMLSC assay development and implementation guidelines.
     
    Documents
  • HCS for Autophagy APAC Document
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    bullet point  MKP-3 Chemical Complementation assay
     
     
    MKP-3 (PYST1) is a dual-specific phosphatase that dephosphorylates MAPK on threonine and tyrosine residues (Groom et al., 1996). The study of MKP biology has thus far been limited to genetic approaches as small molecule inhibitors are lacking. The most popular approach to identifying potential phosphatase inhibitors are in vitro assays using recombinant enzymes small molecule synthetic substrates, such as paranitrophenyl phosphate or O-methyl fluorescein phosphate. These small molecule substrates have been employed because they provide a robust output signal, are inexpensive and are more convenient to use than the phosphorylated protein substrate. Nonetheless, we now know that the activity of MKPs depends on their interaction with other proteins in the cell. This has been documented in great detail for MKP-3, which requires binding to its activated substrate, Erk2, for full enzymatic activity (Camps et al., 1998). Thus, it would seem desirable to develop a method to analyze MKPs in their physiological environment. The availability of a cell active, selective MKP-3 inhibitor would make an immediate impact on the dissection of the complex regulatory processes involved in the attenuation of mitogenic and stress signaling. Selective inhibitors of MKP-3 would also help define the contributions of MKP-3 and its cellular targets to cell growth, death, and organism development. Importantly, an assay for MKP-3 would be an extremely important counterscreen for MKP-1 inhibitors. This is because MKP-1 appears to be a valid anticancer target, whereas although high levels of MKP-3 have been found in cells transformed with the Ret oncogene (Colucci-D'Amato et al., 2000),
    MKP-3 has attributes of a tumor suppressor (reviewed in (Ducruet et al., 2005). Despite the existence of a crystal structure, no potent specific inhibitors of MKP-3 have been reported. Sodium orthovanadate, phenylarsine oxide, and a NSC 357756, a 3-aminoindole derivative (Vogt et al., 2003), are the only inhibitors of MKP-3 described to date. This is partly due to ambiguities and technical challenges in vitro enzyme assays as well as a lack of specific cellular assays for phosphatase inhibition. We have begun to address the problems in phosphatase inhibitor discovery by developing specific assays for phosphatase inhibition in intact mammalian cells based on a “chemical complementation” strategy, which we previously used to prove inhibition of the Cdc25A cell cycle phosphatase in the cell by small molecules (Vogt et al., 2001; Lazo et al., 2001). More recently, we have translated this concept into a single-cell multiparametric assay for MKP-3 activity (Vogt et al., 2003). The inception and development of the Chemical Complementation assay, including its conversion from cell population average format (i.e. Western blot) to a single-cell multiparameter HCS assay have been described in detail (Vogt et al., 2001; Lazo et al., 2001; Vogt et al., 2003; Vogt et al., 2005; Vogt and Lazo, 2005).
     
    Documents
  • MKP-3CC PMLSC APAC Document
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